Hepatic Homeostasis of Metal Ions Following Acute Repeated Stress Exposure in Rats

Essential metals such as copper, iron, and zinc are cofactors in various biological processes including oxygen utilisation, cell growth, and biomolecular synthesis. The homeostasis of these essential metals is carefully controlled through a system of protein transporters involved in the uptake, storage, and secretion. Some metal ions can be transformed by processes including reduction/oxidation (redox) reactions, and correspondingly, the breakdown of metal ion homeostasis can lead to formation of reactive oxygen and nitrogen species. We have previously demonstrated rapid biochemical responses to stress involving alterations in the redox state to generate free radicals and the resultant oxidative stress. However, the effects of stress on redox-active metals including iron and copper and redox-inert zinc have not been well characterised. Therefore, this study aims to examine the changes in these essential metals following exposure to short-term repeated stress, and to further elucidate the alterations in metal homeostasis through expression analysis of different metal transporters. Outbred male Wistar rats were exposed to unrestrained (control), 1 day, or 3 days of 6 h restraint stress (n = 8 per group). After the respective stress treatment, blood and liver samples were collected for the analysis of biometal concentrations and relative gene expression of metal transporter and binding proteins. Exposure to repeated restraint stress was highly effective in causing hepatic redox imbalance. Stress was also shown to induce hepatic metal redistribution, while modulating the mRNA levels of key metal transporters. Overall, this study is the first to characterise the gene expression profile of metal homeostasis following stress and provide insight into the changes occurring prior to the onset of chronic stress conditions.

Alpha2-antiplasmin deficiency affects depression and anxiety-like behavior and apoptosis induced by stress in mice

Objectives Depression is a psychiatric disorder that affects about 10% of the world’s population and is accompanied by anxiety. Depression and anxiety are often caused by various stresses. However, the etiology of depression and anxiety remains unknown. It has been reported that alpha2-antiplasmin (α2AP) not only inhibits plasmin but also has various functions such as cytokine production and cell growth. This study aimed to determine the roles of α2AP on the stress-induced depression and anxiety. Methods We investigated the mild repeated restraint stress-induced depressive and anxiety-like behavior in the α2AP+/+ and α2AP−/− mice using the social interaction test (SIT), sucrose preference test (SPT), and elevated plus maze (EPM). Results The stresses such as the mild repeated restraint stress suppressed α2AP expression in the hippocampus of mice, and the treatment of fluoxetine (selective serotonin reuptake inhibitor [SSRI]) recovered the stress-caused α2AP suppression. We also showed that α2AP deficiency promoted the mild restraint stress-stimulated depression-like behavior such as social withdrawal and apathy and apoptosis in mice. In contrast, α2AP deficiency attenuated the mild restraint stress induced the anxiety-like behavior in mice. Conclusions α2AP affects the pathogenesis of depression and anxiety induced by stress.

Mice cohabiting with familiar conspecific in chronic stress condition exhibit methamphetamine-induced locomotor sensitization and augmented consolation behavior.

Recognize and share emotions are essential for species survival, but in some cases, living with a conspecific in distress condition may induce negative emotional states through empathy-like processes. Studies have reported that stressors promote psychiatric disorders in both, who suffers directly and who witness these aversive episodes, principally whether social proximity is involved. However, the mechanisms underlying the harmful outcomes of emotional contagion needs more studies, mainly in the drug addiction-related behaviors. Here, we investigated the relevance of familiarity and the effects of cohabitation with a partner submitted to chronic stress in the anxiety-like, locomotor sensitization and consolation behaviors. Male swiss mice were housed in pairs during different periods to test the establishment of familiarity and the stress-induced anxiety behavior in the elevated plus maze. Another cohort was housed with a conspecific subjected to repeated restraint stress (1h/day) for 14 days. During chronic restraint the allogrooming was measured and after the stress period mice were tested in the open field for evaluation of anxiety and locomotor cross-sensitization induced by methamphetamine. We found that familiarity was established after 14 days of cohabitation and the anxiogenic behavior appeared after 14 days of stress. Repeated restraint stress also increased anxiety in the open field test and induced locomotor cross-sensitization in the stressed mice and their cagemates. Cagemates also exhibited increase in consolation behavior after stress sessions when compared to control mice. These results indicate that changes in drug abuse-related, consolation and affective behaviors may be precipitate through emotional contagion in familiar conspecifics.

Long-term sensitization of rat spinal neurons induced by adolescent psychophysical stress is further enhanced by a mild-nociceptive lumbar input

Background: Non-specific low back pain (LBP) is one of the most common chronic pain conditions and adverse childhood experiences (ACEs) are known mediators for chronicity of LBP. Sensitization of dorsal horn neurons (DHNs) is a significant element that contributes to chronic LBP. Repeated restraint stress in adult animals is known to cause manifest DHN sensitization when combined with a short-lasting nociceptive input. Objective: In this study, we investigated whether repeated restraint stress in early adolescence leads to a long-term sensitization of DHNs and if an additional mild-nociceptive input by intramuscular nerve growth factor (NGF) leads to further sensitization. Methods: Adolescent Wistar rats were stressed repeatedly in a narrow plastic restrainer, 1 hour per day for 12 consecutive days. Control animals were handled but not restrained. In adulthood, rats were treated with intramuscular injections of saline or NGF (short-lasting mild-nociceptive input) into the lumbar multifidus muscle (L5). Behavioral tests for pain sensitivity were performed before, after stress and inconjunction with intramuscular injections. Rats were transcardially perfused and immunohistochemistry was performed on lumbar (L2) spinal segments. Results: Adolescent restraint stress significantly lowered the low back pressure pain threshold (PPT) immediately after the stress (p<0.0001) and was maintained throughout adulthood (p<0.05). Additionally, paw withdrawal threshold (PWT) was significantly lowered by stress (p<0.0001) but normalized towards adulthood. An NGF injection in adulthood in previously stressed animals lowered the PPT (Cohen d=0.87) and increased microglia marker (Iba-1) immunoreactive area in the superficial DHN (p<0.01) with a trend in increased feret diameter of the immunoreactive cell (p=0.05). Conclusions: Our adolescence stress model induced behavioral signs of sensitization and enhanced sensitivity to further sensitization and dorsal horn microglia activation by subsequent mild nociceptive input (NGF injection). These findings help to understand certain aspects of how adolescent stress might predispose to exacerbation of pain with an additional insult.

Crosstalk Between ATP-P2X7 and Adenosine A2A Receptors Controlling Neuroinflammation in Rats Subject to Repeated Restraint Stress

Depressive conditions precipitated by repeated stress are a major socio-economical burden in Western countries. Previous studies showed that ATP-P2X7 receptors (P2X7R) and adenosine A2A receptors (A2AR) antagonists attenuate behavioral modifications upon exposure to repeated stress. Since it is unknown if these two purinergic modulation systems work independently, we now investigated a putative interplay between P2X7R and A2AR. Adult rats exposed to restraint stress for 14 days displayed an anxious (thigmotaxis, elevated plus maze), depressive (anhedonia, increased immobility), and amnesic (modified Y maze, object displacement) profile, together with increased expression of Iba-1 (a marker of microglia “activation”) and interleukin-1β (IL1β) and tumor necrosis factor α (TNFα; proinflammatory cytokines) and an up-regulation of P2X7R (mRNA) and A2AR (receptor binding) in the hippocampus and prefrontal cortex. All these features were attenuated by the P2X7R-preferring antagonist brilliant blue G (BBG, 45 mg/kg, i.p.) or by caffeine (0.3 g/L, p.o.), which affords neuroprotection through A2AR blockade. Notably, BBG attenuated A2AR upregulation and caffeine attenuated P2X7R upregulation. In microglial N9 cells, the P2X7R agonist BzATP (100 μM) or the A2AR agonist CGS26180 (100 nM) increased calcium levels, which was abrogated by the P2X7R antagonist JNJ47965567 (1 μM) and by the A2AR antagonist SCH58261 (50 nM), respectively; notably JNJ47965567 prevented the effect of CGS21680 and the effect of BzATP was attenuated by SCH58261 and increased by CGS21680. These results provide the first demonstration of a functional interaction between P2X7R and A2AR controlling microglia reactivity likely involved in behavioral adaptive responses to stress and are illustrative of a cooperation between the two arms of the purinergic system in the control of brain function.

De novo protein synthesis is necessary for priming in preclinical models of migraine

Background Migraine attacks are often triggered by normally innocuous stimuli, suggesting that sensitization within the nervous system is present. One mechanism that may contribute to neuronal sensitization in this context is translation regulation of new protein synthesis. The goal of this study was to determine whether protein synthesis contributes to behavioral responses and priming in preclinical models of migraine. Methods Mice received a dural injection of interleukin-6 in the absence or presence of the protein synthesis inhibitor anisomycin or the translation initiation inhibitor 4EGI-1 and were tested for facial hypersensitivity. Upon returning to baseline, mice were given a second, non-noxious dural injection of pH 7.0 to test for priming. Additionally, eIF4ES209Amice lacking phosphorylation of mRNA cap-binding protein eIF4E received dural interleukin-6 or were subjected to repeated restraint stress and then tested for facial hypersensitivity. After returning to baseline, mice were given either dural pH 7.0 or a systemic sub-threshold dose of the nitric oxide donor sodium nitroprusside and tested for priming. Results Dural injection of interleukin-6 in the presence of anisomycin or 4EGI-1 or in eIF4ES209Amice resulted in the partial attenuation of acute facial hypersensitivity and complete block of hyperalgesic priming. Additionally, hyperalgesic priming following repeated restraint stress was blocked in eIF4ES209Amice. Conclusions These studies show that de novo protein synthesis regulated by activity-dependent translation is critical to the development of priming in two preclinical models of migraine. This suggests that targeting the regulation of protein synthesis may be a novel approach for new migraine treatment strategies.