Changes in iNOS,GFAP and NR1 Expression in Various Brain Regions and Elevation of Sphingosine-1-phosphate in Serum after Immobilized Stress

Several studies have been suggested that long-term exposure to stress has detrimental effects on various brain functions and leads to neurodegenerative changes. However, the precise mechanism by which stress induces brain damage or neurodegenerative change is still a matter of debate. This study investigated the damage of neuronal cells involving in the expression of iNOS, NR1, and GFAP in various brain regions and characterized the change of sphingolipid metabolites as a biomarker of physiological change in serum after 3 weeks of repeated immobilization. In this report, the expression of iNOS, GFAP and NR1 in the brain of rats exposed to chronic immobilization stress was investigated. The expression of iNOS, GFAP and NR1 was elevated in the cortex and hippocampal area after 3 weeks of repeated immobilization. Immunoreactivity for GFAP and vimentin, as a marker of reactive gliosis, was also elevated in the cortex and hippocampus. The level of sphingolipids was measured in order to assess the changes in sphingolipid metabolites in the serum of rats exposed to stress. Interestingly, the level of So-1-P was increased in the plasma of rats subjected to 6-h immobilization stress than repeated immobilization. To further investigate the modulating effect of increased So-1-P in various brain regions, So-1-P was infused into the lateral cerebroventricle at a rate of 100 pmol/10 μl/h for 7 days. The expression of iNOS and NR1 was elevated in the cortex, hippocampus, striatum, and cerebellum after So-1-P infusion into the cerebroventricle, while the level of GFAP was elevated in the hippocampus and striatum. Interestingly, the expression levels of iNOS, GFAP, and NR1 were increased by the direct application of So-1-P to cultured cortical cells. These results suggest that NO production via iNOS expression, the NR1 expression, the activation of astrocytes, and the elevation of So-1-P may cause neurodegenerative changes in rats subjected to chronic immobilization and that the elevation of So-1-P by stress exposure would be one of the stress signal molecules.     

Cytochemical Alterations in the Rat Retina by LPS Administration

LPS-induced inflammation and changes in protein phosphorylation and the JAK-STAT pathway accompanying glial activation after LPS treatment, were followed by analyzing secreted proinflammatory cytokine levels. The administration of LPS caused tyrosine phosphorylation of STAT3 in retinae and induced glial fibrillary acidic protein. (GFAP) from the nerve fiber layer to the ganglion cell layer. Our results suggest that the LPS-induced activation of the JAK2/STAT3 signaling pathway may play a key role in the induction of astrogliosis. However, no significant increase in vimentin, OX-42 or inducible nitric oxide synthase (iNOS) expressions were observed after LPS administration. Sphingosine kinase catalyzes the conversion of sphingosine to sphingosine-1–phosphate (So-1-P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, it was found that sphingolipid metabolite levels were elevated in the serum and retinae of LPS-injected rats. To further investigate the chronic effect of increased So-1-P in the retina, So-1-P was infused intracerebroventricularly (i.c.v.) into rats using an osmotic minipump at 100 pmol/10 μl h^-1 for 7 days, and was found to increase retinal GFAP expression. These observations suggest that LPS induces the activation of retinal astrocytes via JAK2/STAT3 and that LPS affects So-1-P generation. Our findings also suggest that elevated So-1-P in the retina and/or in serum could induce cytochemical alterations in LPS treated or inflamed retinae.     

Evidence of lasting dysregulation of neuroendocrine and HPA axis function following global cerebral ischemia in male rats and the effect of Antalarmin on plasma corticosterone level

Abnormal function of the neuroendocrine stress system has been implicated in the behavioral impairments observed following brain ischemia. The current study examined long-term changes in stress signal regulation 30 days following global cerebral ischemia. Experiment 1 investigated changes in the expression of corticotropin releasing hormone (CRH) and its subtype 1 receptor (CRHR1), glucocorticoid receptors (GR) in the paraventricular nucleus of the hypothalamus (PVN), the central nucleus of the amygdala (CeA), and the CA1 subfield of the hippocampus. Tyrosine hydroxylase (TH) was determined at the locus coeruleus (LC). Experiment 2 investigated the role of central CRHR1 activation on corticosterone (CORT) secretion at multiple time intervals following global ischemia after exposure to an acute stressor. Findings from Experiment 1 demonstrated a persistent increase in GR, CRH and CRHR1 immunoreactivity (ir) at the PVN, reduced GR and CRHR1 expression in pyramidal CA1 neurons, and increased LC TH expression in ischemic rats displaying working memory errors in the radial arm Maze. Findings from Experiment 2 revealed increased CORT secretion up to 7 days, but no longer present 14 and 21 days post ischemia. However upon an acute restraint stress induced 27 days following reperfusion, ischemic rats had increased plasma CORT secretions compared to sham-operated animals, suggesting HPA axis hypersensitivity. Antalarmin (2 μg/2 μl) pretreatment significantly attenuated post ischemic elevation of basal and stress-induced CORT secretion. These findings support persistent neuroendocrine dysfunctions following brain ischemia likely to contribute to emotional and cognitive impairments observed in survivors of cardiac arrest and stroke.     

Blockade of TrkB receptors in the nucleus accumbens prior to heterotypic stress alters corticotropin-releasing hormone (CRH), vesicular glutamate transporter 2 (vGluT2) and glucocorticoid receptor (GR) within the mesolimbic pathway

Inhibition of stress-induced elevations in brain-derived neurotrophic factor (BDNF) or its primary receptor tyrosine-related kinase B (TrkB) within the reward pathway may modulate vulnerability to anxiety and mood disorders. The current study examined the role of BDNF/TrkB signaling on biochemistry and behavior under basal conditions and following exposure to a 10-day heterotypic stress paradigm in male rats. Effects of intra-accumbal administration of TrkB antagonist ANA-12 (0.25 μg/0.5 μl/min) on anxiety, and expression of Trk-B, corticotropin-releasing hormone (CRH), vesicular glutamate transporter 2 (vGluT2) and glucocorticoid receptor (GR) within the mesolimbic pathway were determined. Notably, ANA-12 attenuated anxiety-like behavior in stress rats while increasing anxiety in the non-stress group in the elevated plus maze (EPM). At the neurochemical level, ANA-12 blocked the increased vGluT2 and CRH expressions in the hypothalamic PVN and basolateral amygdala in stress rats, while it enhanced vGluT2 and CRH expressions in non-stress rats. ANA-12 also showed state-dependent effects at the NAc core, attenuating TrkB-ir in non-stress rats while reversing reduced expression in stressed rats. At the cingulate cortex, ANA-12 normalized stress-induced increase in TrkB expression. Notably, ANA-12 showed region-specific effects on GR-ir at the NAc core and shell, with increased GR-ir in non-stress rats, although the drug attenuated stress-induced GR-ir expression only in the core portion of the NAc, while having no impact at the cingulate cortex. Elevated blood CORT levels post-stress was not influenced by ANA-12 treatment. Together, these findings suggest that BDNF-mediated TrkB activation exerts differential impact in regulating emotional response under basal and stress conditions.     

Determination of Fecal Glucocorticoid Metabolites to Evaluate Stress Response in <Emphasis Type="Italic">Alouatta pigra</Emphasis>

Measuring fecal glucocorticoid metabolites is now a common practice to assess the stress response in primates. Nevertheless, it is important to validate the utilized immunoassay for each primate species before the technique is applied to populations in the wild. We determined the stress response of black howlers (Alouatta pigra) via 2 different group-specific enzyme immunoassays (EIAs). 11-oxoetiocholanolone EIAs are suited to assess the stress response of black howlers via fecal glucocorticoid metabolites. Levels of fecal glucocorticoid metabolites increased after we applied a stressor, i.e. anesthesia, reaching peak concentrations 24–96 h poststressor. Both basal and stress-induced fecal glucocorticoid metabolite levels showed individual variations. The increase of fecal glucocorticoid metabolites after the stressor (paralleling increases in serum) indicates that one can effectively measure adrenocortical activity in Alouatta pigra via these 2 enzyme immunoassays. However, it is important to consider individual variations in the excretion of fecal glucocorticoid metabolites when planning field endocrinological research on Alouatta pigra. Fecal glucocorticoid metabolite excretion takes 1–3 d poststressor depending on the individual. Further, there is an important individual variability in the concentrations of glucocorticoid metabolites, which might reflect differences in stress reactivity or fecal glucocorticoid metabolite metabolism and excretion.     

Altered stress responsiveness and hypothalamic‐pituitary‐adrenal axis function in male rat offspring of socially isolated parents

Social isolation in male rats at weaning results in reduced basal levels of the neuroactive steroid 3α,5α‐tetrahydroprogesterone (3α,5α‐TH PROG) in the brain and plasma as well as increased anxiety‐like behavior. We now show that socially isolated female rats also manifest a reduced basal cerebrocortical concentration of 3α,5α‐TH PROG as well as an anxiety‐like profile in the elevated plus‐maze and Vogel conflict tests compared with group‐housed controls. In contrast, despite the fact that they were raised under normal conditions, adult male offspring of male and female rats subjected to social isolation before mating exhibited an increased basal cerebrocortical level of 3α,5α‐TH PROG but no difference in emotional reactivity compared with the offspring of group‐housed parents. These animals also showed an increased basal activity of the hypothalamic‐pituitary‐adrenal axis as well as reduced abundance of corticotropin‐releasing factor in the hypothalamus and of corticotropin‐releasing factor receptor type 1 in the pituitary. Moreover, negative feedback regulation of hypothalamic‐pituitary‐adrenal axis activity by glucocorticoid was enhanced in association with up‐regulation of glucocorticoid receptor expression in the hippocampus. There was also attenuation of corticosterone release induced by foot‐shock stress in the offspring of socially isolated parents. The increase in the brain concentration of 3α,5α‐TH PROG induced by acute stress was also blunted in these animals. Our results thus show that a stressful experience before mating can influence neuroendocrine signaling in the next generation.     

Protective effects of therapeutic hypothermia on renal injury in an asphyxial cardiac arrest rat model

Cardiac arrest (CA) is a leading cause of mortality worldwide. Most of post-resuscitation related deaths are due to post-cardiac arrest syndrome (PCAS). After cardiopulmonary resuscitation (CPR), return of spontaneous circulation (ROSC) leads to renal ischemia-reperfusion injury, also known as PCAS. Many studies have focused on brain and heart injuries after ROSC, but renal failure has largely been ignored. Therefore, we investigated the protective effects of therapeutic hypothermia (TH) on asphyxial CA-induced renal injury in rats.Thirty rats were randomly divided into five groups: 1) the control group (sham); 2) the normothermic CA (nor.); 3) a normothermic CA group that received TH immediately within 2 h after CPR (Hypo. 2 hrs); 4) a normothermic CA group that received TH within 4 h after CPR (Hypo. 4 hrs); and 5) a normothermia CA group that received TH within 6 h after CPR (Hypo. 6 h). One day after CPR, all rats were sacrificed. Compared with the normothermic CA group, the TH groups demonstrated significantly increased survival rate (P < 0.05); decreased serum blood urea nitrogen, creatinine, and lactate dehydrogenase levels; and lower histological damage degree and malondialdehyde concentration in their renal tissue. Terminal deoxynucleotidyl transferase dUTP nick end labeling stain revealed that the number of apoptotic cells significantly decreased after 4 h and 6 h of TH compared to the results seen in the normothermic CA group. Moreover, TH downregulated the expression of cyclooxygenase-2 in the renal cortex compared to the normothermic CA group one day after CPR. These results suggest that TH exerts anti-apoptotic, anti-inflammatory, and anti-oxidative effects immediately after ROSC that protect against renal injury.     

Long-Term Behavioral Programming Induced by Peripuberty Stress in Rats Is Accompanied by GABAergic-Related Alterations in the Amygdala

Stress during childhood and adolescence is a risk factor for psychopathology. Alterations in γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain, have been found following stress exposure and fear experiences and are often implicated in anxiety and mood disorders. Abnormal amygdala functioning has also been detected following stress exposure and is also implicated in anxiety and social disorders. However, the amygdala is not a unitary structure; it includes several nuclei with different functions and little is known on the potential differences the impact of early life stress may have on this system within different amygdaloid nuclei. We aimed here to evaluate potential regional differences in the expression of GABAergic-related markers across several amygdaloid nuclei in adult rats subjected to a peripuberty stress protocol that leads to enhanced basal amygdala activity and psychopathological behaviors. More specifically, we investigated the protein expression levels of glutamic acid decarboxylase (GAD; the principal synthesizing enzyme of GABA) and of GABA-A receptor subunits α2 and α3. We found reduced GAD and GABA-A α3, but not α2, subunit protein levels throughout all the amygdala nuclei examined (lateral, basolateral, basomedial, medial and central) and increased anxiety-like behaviors and reduced sociability in peripubertally stressed animals. Our results identify an enduring inhibition of the GABAergic system across the amygdala following exposure to early adversity. They also highlight the suitability of the peripuberty stress model to investigate the link between treatments targeting the dysfunctional GABAergic system in specific amygdala nuclei and recovery of specific stress-induced behavioral dysfunctions.     

Functional Programming of the Autonomic Nervous System by Early Life Immune Exposure: Implications for Anxiety

Neonatal exposure of rodents to an immune challenge alters a variety of behavioural and physiological parameters in adulthood. In particular, neonatal lipopolysaccharide (LPS; 0.05 mg/kg, i.p.) exposure produces robust increases in anxiety-like behaviour, accompanied by persistent changes in hypothalamic-pituitary-adrenal (HPA) axis functioning. Altered autonomic nervous system (ANS) activity is an important physiological contributor to the generation of anxiety. Here we examined the long term effects of neonatal LPS exposure on ANS function and the associated changes in neuroendocrine and behavioural indices. ANS function in Wistar rats, neonatally treated with LPS, was assessed via analysis of tyrosine hydroxylase (TH) in the adrenal glands on postnatal days (PNDs) 50 and 85, and via plethysmographic assessment of adult respiratory rate in response to mild stress (acoustic and light stimuli). Expression of genes implicated in regulation of autonomic and endocrine activity in the relevant brain areas was also examined. Neonatal LPS exposure produced an increase in TH phosphorylation and activity at both PNDs 50 and 85. In adulthood, LPS-treated rats responded with increased respiratory rates to the lower intensities of stimuli, indicative of increased autonomic arousal. These changes were associated with increases in anxiety-like behaviours and HPA axis activity, alongside altered expression of the GABA-A receptor α2 subunit, CRH receptor type 1, CRH binding protein, and glucocorticoid receptor mRNA levels in the prefrontal cortex, hippocampus and hypothalamus. The current findings suggest that in addition to the commonly reported alterations in HPA axis functioning, neonatal LPS challenge is associated with a persistent change in ANS activity, associated with, and potentially contributing to, the anxiety-like phenotype. The findings of this study reflect the importance of changes in the perinatal microbial environment on the ontogeny of physiological processes.     

Maternal Separation Enhances Conditioned Fear and Decreases the mRNA Levels of the Neurotensin Receptor 1 Gene with Hypermethylation of This Gene in the Rat Amygdala

Stress during postnatal development is associated with an increased risk for depression, anxiety disorders, and substance abuse later in life, almost as if mental illness is able to be programed by early life stressors. Recent studies suggest that such “programmed” effects can be caused by epigenetic regulation. With respect to conditioned fear, previous studies have indicated that early life stress influences its development in adulthood, whereas no potential role of epigenetic regulation has been reported. Neurotensin (NTS) is an endogenous neuropeptide that has receptors densely located in the amygdala and hippocampus. Recently, NTS systems have constituted an emerging target for the treatment of anxiety. The aim of the present work is to clarify whether the NTS system is involved in the disturbance of conditioned fear in rats stressed by maternal separation (MS). The results showed that MS enhanced freezing behaviors in fear-conditioned stress and reduced the gene expression of NTS receptor (NTSR) 1 but not of NTS or NTSR2 in the amygdalas of adult rats. The microinjection of a NTSR1 antagonist into the amygdala increased the percentage of freezing in conditioned fear, whereas the microinjection of NTSR1 agonist decreased freezing. These results suggest that NTSR1 in the amygdala may play a role in the effects of MS on conditioned fear stress in adult rats. Moreover, MS increased DNA methylation in the promoter region of NTSR1 in the amygdala. Taken together, MS may leave epigenetic marks in the NTSR1 gene in the amygdala, which may enhance conditioned fear in adulthood. The MS-induced alternations of DNA methylation in the promoter region of NTSR1 in the amygdala may be associated with vulnerability to the development of anxiety disorders and depression in adulthood.     

The effects of acute corticosterone administration on anxiety, endogenous corticosterone, and c-Fos expression in the rat brain

The effects of acute pretreatment of rats with corticosterone (5 and 20 mg/kg, s.c.) on emotional behavior, expression of c-Fos protein in brain structures, and serum concentration of corticosterone were studied to model the short-term glucocorticoid-dependent changes in brain functions. Corticosterone was administered 90 min before training of a conditioned fear reaction (a freezing response), and behavioral, hormonal and immunocytochemical effects were examined 1 day later, on the test day. Pretreatment of rats with corticosterone significantly attenuated the freezing reaction in the conditioned fear test. The effect of the corticosterone was accompanied by a selective enhancement of the aversive context-induced c-Fos expression in some brain structures: the parvocellular and magnocellular neurons of the paraventricular hypothalamic nucleus (pPVN and mPVN), the medial amygdala nucleus (MeA), and the cingulate cortex, area 1 (Cg1), as well as an increase in the concentration of aversive context-induced endogenous serum glucocorticoid, 1.5 h and 10 min after the test session, respectively. It is suggested that the behavioral effects of acute pretreatment of rats with corticosterone could be due to changes in the mnemonic processes in the brain, inhibition of brain corticotropin releasing factor (CRF) synthesis, or stimulation of GABA-A receptor modulating neurosteroids synthesis. It is hypothesized that the enhanced activity of Cg1, MeA, pPVN, and mPVN, and the hypothalamic-pituitary-adrenal axis with concomitant increased serum glucocorticoid concentration, might serve to facilitate active coping behavior in a threatening situation.     

Effects of a high-salt diet on adipocyte glucocorticoid receptor and 11-β hydroxysteroid dehydrogenase 1 in salt-sensitive hypertensive rats

High-salt diets decrease insulin sensitivity in salt-sensitive hypertensive rats, and glucocorticoids promote adipocyte growth and may have pathophysiological roles in the metabolic syndrome. The aim of this study was to clarify the relationship between high-salt diet and the adipocyte glucocorticoid hormones in salt-sensitive hypertensive rats. Six-week-old Dahl salt-sensitive (DS) hypertensive rats and salt-resistant (DR) rats were fed a high-salt diet or a normal-salt diet for 4 weeks. Fasting blood glucose (FBG), serum adiponectin, plasma insulin, and corticosterone in plasma and in visceral adipose tissues, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activities in adipose tissues and glucose uptake in isolated muscle were measured. Animals underwent an oral glucose tolerance test (OGTT). The expression of mRNA for glucocorticoid receptor (GR), 11β-HSD1 and tumor necrosis factor-α (TNF-α) in adipose tissues were measured using a real-time PCR. A high-salt diet did not influence FBG; however, decreased 2-deoxy glucose uptake and plasma insulin during OGTT in DS rats. The high-salt diet increased significantly adipose tissue corticosterone concentration and 11β-HSD1 activities, gene expression for GR, 11β-HSD1 and TNF-α in adipose tissues in DS rats compared with DR rats (p < 0.05). The high-salt diet did not influence plasma corticosterone and serum adiponectin concentration in DS and DR rats. These results suggest that changes in GR and 11β-HSD1 in adipose tissue may contribute to insulin sensitivity in salt-sensitive hypertensive rats.     

Prenatal Morphine Exposure Differentially Alters Addictive and Emotional Behavior in Adolescent and Adult Rats in a Sex-Specific Manner

The effects of prenatal opioid exposure in adult animals has been widely studied, but little is known about the effects of prenatal opioid on adolescents. Most of the risk behaviors associated with drug abuse are initiated during adolescence. The developmental state of the adolescent brain makes it vulnerable to initiate drug use and susceptible to drug-induced brain changes. In this study, pregnant rats were subcutaneously injected with an increasing dose of morphine (5 mg/kg, 7 mg/kg, 10 mg/kg) for 9 days since the gestation day 11. The effects of prenatal morphine (PNM) on learning and memory, anxiety- and depressive- like behavior, morphine induced conditioned place preference (CPP) as well as locomotor sensitization were tested in both adolescent and adult rats. The results showed that: (1) PNM decreased anxiety-like behavior in both adolescent and adult female rats, but not males; (2) PNM decreased depressive-like behavior in adolescent but increased depressive -like behavior in adult females; (3) PNM increased low dose morphine induced locomotor sensitization in females; (4) PNM decreased tyrosine hydroxylase (TH) expression in the prefrontal cortex but decreased dopamine D1 receptor expression in the nucleus-accumbens (NAc) in female rats. These results suggested that PNM altered the emotional and addictive behavior mainly in female rats, with female rats being less anxiety and depressive during adolescence, but more depressive in adult, and more sensitive to low dose morphine induced locomotor activity sensitization, which might be mediated in part by the differential expression of the TH, dopamine D1 receptors in the female brain.

     

Effects of chronic mild stress on behavioral and neurobiological parameters — Role of glucocorticoid

Major depression is thought to originate from maladaptation to adverse events, particularly when impairments occur in mood-related brain regions. Hypothalamus–pituitary–adrenal (HPA) axis is one of the major systems involved in physiological stress response. HPA axis dysfunction and high glucocorticoid concentrations play an important role in the pathogenesis of depression. In addition, astrocytic disability and dysfunction of neurotrophin brain-derived neurotrophin factor (BDNF) greatly influence the development of depression and anxiety disorders. Therefore, we investigated whether depressive-like and anxiety-like behaviors manifest in the absence of glucocorticoid production and circulation in adrenalectomized (ADX) rats after chronic mild stress (CMS) exposure and its potential molecular mechanisms. The results demonstrate that glucocorticoid-controlled rats showed anxiety-like behaviors but not depression-like behaviors after CMS. Molecular and cellular changes included the decreased BDNF in the hippocampus, astrocytic dysfunction with connexin43 (cx43) decreasing and abnormality in gap junction in prefrontal cortex (PFC). Interestingly, we did not find any changes in glucocorticoid receptor (GR) or its chaperone protein FK506 binding protein 51 (FKBP5) expression in the hippocampus or PFC in ADX rats subjected to CMS. In conclusion, the production and circulation of glucocorticoids are one of the contributing factors in the development of depression-like behaviors in response to CMS. In contrast, the effects of CMS on anxiety-like behaviors are independent of the presence of circulating glucocorticoids. Meanwhile, stress decreased GR expression and enhanced FKBP5 expression via higher glucocorticoid exposure. Gap junction dysfunction and changes in BDNF may be associated with anxiety-like behaviors.     

Activation of Different Neuronal Phenotypes in the Rat Brain Induced by Liver Ischemia–Reperfusion Injury: Dual Fos/Neuropeptide Immunohistochemistry

The aim of the present study was to reveal the effect of liver ischemia–reperfusion injury (LIRI) on the activity of selected neuronal phenotypes in rat brain by applying dual Fos-oxytocin (OXY), vasopressin (AVP), tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), corticoliberine (CRH), and neuropeptide Y (NPY) immunohistochemistry. Two liver ischemia–reperfusion models were investigated: (i) single ligation of the hepatic artery (LIRIa) for 30 min and (ii) combined ligation of the portal triad (the common hepatic artery, portal vein, and common bile duct) (LIRIb) for 15 min. The animals were killed 90 min, 5 h, and 24 h after reperfusion. Intact and sham operated rats served as controls. As indicated by semiquantitative estimation, increases in the number of Fos-positive cells mainly occurred 90 min after both liver reperfusion injuries, including activation of AVP and OXY perikarya in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and TH, NPY, and PNMT perikarya in the catecholaminergic ventrolateral medullar A1/C1 area. Moreover, only PNMT perikarya located in the A1/C1 cell group exhibited increased Fos expression 5 h after LIRIb reperfusion. No or very low Fos expression was found 24 h after reperfusion in neuronal phenotypes studied. Our results show that both models of the LIRI activate, almost by the same effectiveness, a number of different neuronal phenotypes which stimulation may be associated with a complex of physiological responses induced by (1) surgery (NPY, TH, PNMT), (2) hemodynamic changes (AVP, OXY, TH, PNMT), (3) inflammation evoked by ischemia and subsequent reperfusion (TH), and (4) glucoprivation induced by fasting (NPY, PNMT, TH). All these events may contribute by different strength to the development of pathological alterations occurring during the liver ischemia–reperfusion injury.