Serelaxin activates eNOS, suppresses inflammation, attenuates developmental delay and improves cognitive functions of neonatal rats after germinal matrix hemorrhage.

Germinal matrix hemorrhage (GMH) is a detrimental form of neonatal CNS injury. Following GMH-mediated eNOS inhibition, inflammation arises, contributing to GMH-induced brain injury. We investigated the beneficial effects of Serelaxin, a clinical tested recombinant Relaxin-2 protein, on brain injury after GMH in rats. We investigated whether effects of Serelaxin are mediated by its ability to activate the GMH-suppressed eNOS pathway resulting in attenuation of inflammatory marker overproduction. GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U). Seven day old Sprague-Dawley rat pups (P7) were used (n = 63). GMH animals were divided in vehicle or serelaxin treated (3 µg once, 30 µg once, 30 µg multiple, i.p., starting 30 after GMH and then daily). Sham operated animals were used. We monitored the developmental profile working memory and spatial function (T-maze and open field test respectively). At day 28, all rats underwent MRI-scans for assessment of changes in cortical thickness and white matter loss. Effects of Serelaxin on eNOS pathway activation and post-GMH inflammation were evaluated. We demonstrated that Serelaxin dose-dependently attenuated GMH-induced developmental delay, protected brain and improved cognitive functions of rats after GMH. That was associated with the decreased post-GMH inflammation, mediated at least partly by amelioration of GMH-induced eNOS inhibition.

Individual differences in inflammatory and oxidative mechanisms of stress-related mood disorders.

Emotional stress leads to the development of peripheral disorders and is recognized as a modifiable risk factor for psychiatric disorders, particularly depression and anxiety. However, not all individuals develop the negative consequences of emotional stress due to different stress coping strategies and resilience to stressful stimuli. In this review, we discuss individual differences in coping styles and the potential mechanisms that contribute to individual vulnerability to stress, such as parameters of the immune system and oxidative state. Initial differences in inflammatory and oxidative processes determine resistance to stress and stress-related disorders via the alteration of neurotransmitter content in the brain and biological fluids. Differences in coping styles may serve as possible predictors of resistance to stress and stress-related disorders, even before stressful conditions. The investigation of natural variabilities in stress resilience may allow the development of new methods for preventive medicine and the personalized treatment of stress-related conditions.

[DYNAMICS OF CYTOKINE CONCENTRATION IN THE BLOOD OF RATS WITH VARIOUS BEHAVIORAL CHARACTERISTICS AFTER ACUTE EMOTIONAL STRESS].

This work was designed to study a change in cytokine content in the peripheral blood of behaviorally passive and active Wistar rats at various time intervals after acute stress on the model of night-time immobilization. A decrease in the concentration of most pro- and anti-inflammatory cytokines in passive animals was most pronounced immediately and, particularly, 3 days after stress exposure. Variations in the blood cytokine profile after experimental stress were lower in behaviorally active specimens. A statistically significant decrease was observed only in the amount of a proinflammatory cytokine IL-1α and anti-inflammatory cytokines IL-4 and IL-13. As differentiated from passive rats, these changes in active specimens were most pronounced 1 day after negative emotiogenic exposure. Our results illustrate a specific involvement of immunoactive substances in the systemic regulation of physiological functions and development of individual resistance to the negative consequences of stress.

[Effect of melatonin on antioxidant enzyme activities in blood erythrocytes of rats during acute emotional stress].

The effect of the epiphyseal hormone melatonin on the activity of antioxidant enzymes, glutathione peroxidase (GPx), glutathione reductase (GR), and Cu/Zn-superoxide dismutase (Cu/Zn-SOD) was studied in peripheral blood erythrocytes of behaviorally passive and active Wistar rats. Acute emotional stress was modeled by immobilization of animals for1 h with simultaneous electrocutaneous stimulation. Basal activity of antioxidant glutathione enzymes in erythrocytes of behaviorally passive rats was higher than that in active animals. Administration of melatonin (2 mg/kg, intraperitoneally) was accompanied by a decrease in the activity of GPx and GR in erythrocytes from non-stressed passive animals. After experimental stress, passive rats demonstrated a significant increase in the activity of Cu/Zn-SOD and GPx in peripheral blood erythrocytes. The absence of stress-induced changes in functional activity of antioxidant defense enzymes in the blood of behaviorally active animals suggests a relatively constant oxidative status of tissues in these animals under stress conditions. Melatonin administration had little effect on stress-induced changes in functional activity of the erythrocyte antioxidant system in passive rats. Active specimens pretreated with melatonin before stress exposure were characterized by activation of study antioxidant enzymes. Quantitative parameters of the erythrocyte antioxidant defense enzymes did not differ in behaviorally active and passive rats subjected to experimental stress after melatonin injection. Thus, exogenous melatonin abolishes differences in the activity of study antioxidant enzymes in erythrocytes of animals with different behavioral parameters under basal conditions and after experimental stress. In passive rats melatonin mainly reduced the initial tension of oxidative processes. By contrast, administration of this hormone to active specimens is followed by an increase in functional activity of the antioxidant enzyme system under conditions of acute stress.

Effect of interleukin-4 on peripheral blood leukocytes in rats with various behavioral characteristics during acute stress.

We studied the effect of anti-inflammatory cytokine IL-4 (5 µg/kg intraperitoneally) on peripheral blood leukocytes in Wistar rats with various behavioral characteristics during acute emotional stress (1-h immobilization with simultaneous subthreshold electrocutaneous stimulation). IL-4 reduced the differences in blood leukocyte count in rats with various behavioral characteristics, which was related to a significant decrease in this parameter in active animals. IL-4 injection to active animals was accompanied by changes in the leukogram (development of neutrophilia, monocytopenia, and lymphopenia) and had a modulatory effect on leukocyte indexes of cell reactivity. Blood leukocyte count in cytokine-treated animals did not change after stress exposure. IL-4 prevented shifts in leukocyte indexes of cell reactivity, which was found after acute stress exposure. Our results expand current notions on the specific involvement of endogenous immunomodulatory compounds in the realization of adaptive and compensatory processes in mammals during negative emotiogenic exposures.

Proteomic study of rat hippocampus under conditions of emotional stress.

Proteomic differences in the hippocampus of stress-resistant and stress-sensitive rats were detected on the model of emotional stress. Differential expression of some proteins was detected in animals with different behavioral activity initially and after experimental stress exposure. Specific involvement of the hippocampus in the realization of stress response in animals with different sensitivity to emotional stress was demonstrated.

Melatonin effects on serum cytokine profiles of rats with different behavioral parameters in acute emotional stress.

The effects of melatonin (epiphyseal neurohormone) on the serum cytokine profiles of rats with different behavioral characteristics were studied after acute emotional stress. One-hour immobilization of animals with simultaneous electrocutaneous stimulation of subthreshold intensity served as the stress model. Acute stress exposure of animals with active behavior led to reduction of the peripheral blood concentrations of pro-inflammatory (IL-1α, IL-1ß, IL-2, IFN-γ, granulomonocytic CSF) and anti-inflammatory (IL-4, IL-10) cytokines. Passive rats exposed to emotional stress developed a pronounced increase of pro-inflammatory IL-1ß concentration. Reduction of pro-inflammatory cytokine levels in active rats exposed to stress was less pronounced after intraperitoneal preinjection of melatonin (2 mg/kg). In passive animals, exogenous melatonin inverted the poststress changes in the serum levels of pro-inflammatory IL-2 cytokine and of anti-inflammatory IL-4 and IL-10 cytokines. The modulatory effect of melatonin on the cytokine profiles of rats with different behavioral parameters seemed to contribute to adaptation of animals to emotional stress exposure.

Effect of interleukin-1ß on peripheral blood leukocytes in rats with various behavioral characteristics during acute stress.

We studied the effect of a pro-inflammatory cytokine IL-1ß (5 µg/kg intraperitoneally) on blood leukocytes in Wistar rats various behavioral characteristics during acute emotional stress (1-h immobilization with simultaneous delivery of subthreshold electrocutaneous stimulation). Stress exposure was accompanied by a decrease in the total number of peripheral blood leukocytes in rats. Active animals were characterized by the increase in neutrophil count during stress. The number of eosinophils in passive specimens was shown to decrease under these conditions. Emotional stress was followed by a decrease in the lymphocyte index (by Shaganin) of active rats and increase in the leukocyte intoxication index (according to Kalf-Kalif) of passive specimens. Stress-induced changes in leukocytes differed after pretreatment with IL-1ß. The number of blood leukocytes increased in animals receiving a cytokine injection before stress exposure. Exogenous IL-1ß inverted (in active rats) or prevented (in passive specimens) a change in the percentage of various types of blood leukocytes, which was found after stress exposure. These data contribute to the understanding of peripheral mechanisms for the involvement of immunomodulatory cytokines in the systemic organization of physiological functions in specimens with different prognostic resistance to a similar stress exposure.

Cytokine profile of peripheral blood in rats with various behavioral characteristics during acute emotional stress.

The cytokine profile of peripheral blood plasma was studied in rats with various behavioral characteristics after acute emotional stress (1-hour immobilization with simultaneous electrocutaneous stimulation). Under basal conditions the concentrations of pro-inflammatory (IL-1α, IL-1ß, IL-2, IFN-γ, and granulocyte-monocyte CSF) and anti-inflammatory cytokines (IL-4 and IL-10) in the blood of active animals were higher than in passive specimens. Acute stress was accompanied by a decrease in the level of plasma cytokines in behaviorally active rats. Stressed passive specimens were characterized by the accumulation of a pro-inflammatory cytokine IL-1ß and anti-inflammatory cytokine IL-4 in the peripheral blood. The observed differences in the cytokine profile of the blood in behaviorally passive and active rats under basal conditions and after a negative emotiogenic exposure can be related to the specifics of immune reactions and metabolic processes in animals with different prognostic resistance to similar stress factors.

[Effect of melatonin on lipid peroxidation in the blood of rats with various behavioral characteristics during acute emotional stress].

This work was designed to study the effect of melatonin on lipid peroxidation in the peripheral blood of behaviorally passive and active Wistar rats. Immobilization of rats with simultaneous electrocutaneous stimulation (1 h) served as a model of acute stress. After intraperitoneal injection of melatonin (2 mg/kg) the intensity of lipid peroxidation in the blood plasma remained practically unchanged in passive specimens, but increased in active animals. Stress exposure was followed by specific variations in free radical processes in the blood (passive rats, inhibition; active specimens, no changes). Administration of melatonin contributed to a decrease in the intensity of lipid peroxidation in the blood of stressed rats (as compared to control specimens receiving this neurohormone). Therefore, the effect of melatonin on free radical processes depends on the initial behavioral characteristics and physiological state of animals. These data illustrate the importance of an individual approach to studying the systemic mechanisms for organization of functions in mammals. Key words: melatonin, emotional stress, lipid peroxidation, blood plasma, rats with various behavioral characteristics.

Effect of interleukin-4 on antioxidant protection of the brain in rats during acute emotional stress.

We studied the effect of IL-4 on antioxidant enzyme activity in brain structures (hypothalamus, sensorimotor cortex, and amygdala) in behaviorally passive and active rats. One-hour immobilization of animals with simultaneous delivery of subthreshold electrocutaneous stimulation was used as the model of acute stress. Intraperitoneal injection of IL-4 (5 µg/kg) was followed by an increase in activities of glutathione peroxidase and Cu/Zn SOD in the hypothalamus of non-stressed rats. Activities of glutathione peroxidase and Cu/Zn SOD in the amygdala were shown to decrease. Administration of IL-4 was accompanied by activation of glutathione peroxidase (active and passive rats), glutathione reductase (passive rats), and Cu/Zn SOD (active rats) in the sensorimotor cortex. These data indicate that the efficiency of antioxidant protection increases in the hypothalamus and sensorimotor cortex, but decreases in the amygdala of rats receiving IL-4. Pretreatment with IL-4 abolished a poststress increase in glutathione peroxidase activity in the sensorimotor cortex of passive animals.

Cytokine levels in rat blood and brain structures after administration of lipopolysaccharide.

We compared cytokine profile of rat serum and brain structures after immune status modulation by LPS (30 µg/kg intraperitoneally). The content of inflammatory (IL-1α, IL-1ß, IL-2, IL-6, IFN-γ, and TNF-α) and anti-inflammatory (IL-4 and IL-10) cytokines in biological samples of animals was measured on days 1 and 7 after antigenic stimulation. LPS administration reduced the levels of both inflammatory and anti-inflammatory cytokines in the peripheral blood of the rats, especially on the 1st day. LPS administration was also accompanied by specific changes in cytokine content in the dorsal hippocampus and anterior cingulate cortex. Antigenic stimulation increased the level of anti-inflammatory cytokines IL-4 and IL-10 in the examined brain tissues, the changes were most pronounced on day 1 after LPS injection. No significant changes in the levels of proinflammatory cytokines in the brain tissue of animals were found at the above terms after LPS injection. Thus, peripheral LPS administration to rats shifts the balance between the inflammatory and anti-inflammatory cytokines in the CNS structures towards the latter.

Antioxidant protection of the brain in rats during acute stress and administration of interleukin-1ß.

We studied the effect of IL-1ß on antioxidant enzyme activity in emotiogenic structures of the brain (hypothalamus, sensorimotor cortex, and amygdala) in behaviorally passive and active rats with different sensitivity to stress. One-hour immobilization of animals with simultaneous electrocutaneous stimulation was used as a model of stress. An intraperitoneal injection of IL-1ß (5 µg/kg) was followed by the decrease in glutathione reductase activity in the hypothalamus of rats. Behaviorally active animals of the IL-1ß group were characterized by an increase in the activities of Cu/Zn superoxide dismutase and glutathione peroxidase in the sensorimotor cortex and amygdala, respectively. IL-1ß administration was accompanied by activation of Cu/Zn superoxide dismutase and glutathione peroxidase in the amygdala of passive rats. Pretreatment with IL-1ß abolished the poststress changes in enzyme activity in the hypothalamus and sensorimotor cortex of active and passive rats, respectively. These data illustrate the specific effects of IL-1ß on antioxidant protection of CNS tissues in rats with various behavioral characteristics.

[Effect of interleukin-1beta and interleukin-4 on serum albumin parameters in rats with various behavioural characteristics].

We studied the effects of pro-inflammatory cytokine interleukin-1beta and antiinflammatory cytokine interleukin-4 on serum albumin parameters in rats with various behavioural characteristics in the open-field test. Under control conditions, the total concentration of serum albumin in active animals was higher than in passive those. However, the ratio of the effective-to-total concentration of albumin (i.e., binding capacity of this protein) was greater in passive rats. Administration of interleukins was accompanied by a decrease in the total content and effective concentration of albumin in passive and, particularly, in active rats. The initial intergroup differences in the ratio of the effective-to-total concentration of albumin were not found after injection of immunomodulating agents. It was mainly related to a more significant increase in this parameter in active animals. An increase in the binding capacity of albumin after cytokine treatment could be associated with conformational changes in the protein molecule. We believe that interleukin-1b and interleukin-4 exert similar effects on the properties of binding sites of serum albumin in rats with various behavioural characteristics.

Comparative analysis of the effect of cytokines on the thymus, adrenal glands, and spleen in rats with various behavioral characteristics.

We compared the effect of a pro-inflammatory cytokine IL-1ß and anti-inflammatory cytokine IL-4 on the state of stress-marker organs in rats with various behavioral characteristics in the open-field test. Intraperitoneal injection of the cytokines was followed by a slight decrease in the relative weight of the thymus, adrenal glands, and spleen in behaviorally active and, especially, in passive animals. These changes are probably associated with the effect of the test immunomodulators on apoptosis and migration of cells of the immunocompetent organs. Pretreatment with IL-1ß and IL-4 was shown to prevent involution of the thymus and spleen in rats during acute stress on the model of immobilization with simultaneous electrocutaneous stimulation. Hence, these cytokines have the same effects on functional state of stress-marker organs in animals. IL-1ß and IL-4 have a greater effect in passive rats than in active specimens, which reflects specific features of immune mechanisms in animals with different emotional reactivity.

Intensity of oxidative and antioxidant processes in the brain of rats with various behavioral characteristics during acute stress.

We studied the effect of acute emotional stress (1-h immobilization with simultaneous electrocutaneous stimulation) on the prooxidant-antioxidant balance in emotiogenic structures of the brain in rats with various behavioral characteristics. TBA-reactive substance content in the hypothalamus of rats remained practically unchanged after stress exposure. Opposite changes in activity of antioxidant defense enzymes in this structure of the brain in behaviorally active specimens probably compensate for the possible variations in LPO during emotional stress. Activities of glutathione reductase and Cu/Zn-containing SOD in the hypothalamus of passive animals decreased under these conditions. As differentiated from active rats, emotional stress in passive specimens was accompanied by the accumulation of TBA-reactive substances in the sensorimotor cortex and amygdala. The observed increase in glutathione peroxidase activity in passive animals probably serves as a secondary compensatory reaction to LPO activation. Our results illustrate specific changes in free radical processes and antioxidant defense in emotiogenic structures of the brain in rats with various behavioral characteristics after acute stress. These changes were more pronounced in behaviorally passive specimens than in active animals. It was probably related to differences in the oxidative status of CNS in rats with various prognostic resistance to similar stress factors.

Modulating effect of interleukin-4 on free radical processes in the brain of rats during emotional stress.

We studied the effect of anti-inflammatory cytokine IL-4 on the intensity of free radical processes in emotiogenic brain structures (hypothalamus, sensorimotor cortex, and amygdala) in rats with different prognostic emotional resistance. One-hour immobilization of animals with simultaneous electrocutaneous stimulation was used as a model of acute stress. The stress was accompanied by accumulation of MDA (LPO end-product) in the sensorimotor cortex and amygdala of passive rats. Intraperitoneal administration of IL-4 (5 µg/kg) increased MDA content in the amygdala and hypothalamus of non-stressed rats with different behavioral activity. In the sensorimotor cortex of passive and, especially, active rats, a decrease in MDA level was observed after injection of this cytokine. Preliminary administration of IL-4 prevented LPO activation in the sensorimotor cortex of behaviorally passive animals observed after stress against the background of saline injection. Regional peculiarities of LPO under the influence of this cytokine can be determined by differences in both biochemical processes in the brain tissue and specific involvement of different emotiogenic structures in the formation of the stress response. The revealed differences in the effects of IL-4 on free radical processes in active and passive rats indicate peculiarities of immune mechanisms in animals with different resistance to the same type of stress.

Effect of interleukin-1ß on lipid peroxidation in emotiogenic structures of the brain in rats during acute stress.

We studied the effects of immunomodulatory cytokine interleukin-1ß on lipid peroxidation in emotiogenic structures of the brain (hypothalamus, sensorimotor cortex, and amygdala) of behaviorally active and passive rats with different prognostic resistance to stress. Immobilization of animals with simultaneous electrocutaneous stimulation (1 h) served as the model of acute emotional stress. Intraperitoneal injection of IL-1ß (5 µg/kg) was followed by accumulation of malonic dialdehyde (end-product of lipid peroxidation) in all structures of the brain in passive rats, as well as in the hypothalamus of active animals. As differentiated from active rats, stress exposure in passive specimens was accompanied by a selective increase in malonic dialdehyde content in the sensorimotor cortex and amygdala. Pretreatment with IL-1ß prevented activation of lipid peroxidation in the studied structures of the brain in passive rats after stress exposure. Our results show the specific effect of IL-1ß on free-radical processes in the hypothalamus, sensorimotor cortex, and amygdala in rats with various behavioral parameters. Regional features of lipid peroxidation in emotiogenic structures of the brain in animals with different emotional reactivity probably contribute to the existence of significant variations in the individual resistance to emotional stress.

Effect of interleukin-1beta on the behavior of rats during mild stress in the open-field test.

We studied the effect of interleukin-1beta on the behavior of rats with different individual typological characteristics during mild stress in the open-field test. Intraperitoneal injection of interleukin-1beta (5 microg/kg, 108 U/mg) was followed by a decrease in orientation and exploratory activity of passive and, particularly, of active animals in the open field. As differentiated from rats receiving physiological saline, the initial differences in behavioral characteristics of active and passive animals were not revealed in the repeated test after injection of interleukin-1beta. We conclude that interleukin-1beta abolishes the behavioral differences between active and passive specimens in the open field. These data suggest that administration of interleukin-1beta to rats leads to reorganization of the mechanisms for emotional evaluation of adverse emotiogenic factors under conditions of mild stress in the open-field test.