Nerve Injury Triggers Time-dependent Activation of the Locus Coeruleus, Influencing Spontaneous Pain-like Behavior in Rats.

BACKGROUND: Dynamic changes in neuronal activity and in noradrenergic locus coeruleus (LC) projections have been proposed during the transition from acute to chronic pain. Thus, the authors explored the cellular cFos activity of the LC and its projections in conjunction with spontaneous pain-like behavior in neuropathic rats. METHODS: Tyrosine hydroxylase:Cre and wild-type Long-Evans rats, males and females, were subjected to chronic constriction injury (CCI) for 2 (short-term, CCI-ST) or 30 days (long-term, CCI-LT), evaluating cFos and Fluoro-Gold expression in the LC, and its projections to the spinal cord (SC) and rostral anterior cingulate cortex (rACC). These tests were carried out under basal conditions (unstimulated) and after noxious mechanical stimulation. LC activity was evaluated through chemogenetic and pharmacologic approaches, as were its projections, in association with spontaneous pain-like behaviors. RESULTS: CCI-ST enhanced basal cFos expression in the LC and in its projection to the SC, which increased further after noxious stimulation. Similar basal activation was found in the neurons projecting to the rACC, although this was not modified by stimulation. Strong basal cFos expression was found in CCI-LT, specifically in the projection to the rACC, which was again not modified by stimulation. No cFos expression was found in the CCI-LT LCipsilateral (ipsi)/contralateral (contra)→SC. Chemogenetics showed that CCI-ST is associated with greater spontaneous pain-like behavior when the LCipsi is blocked, or by selectively blocking the LCipsi→SC projection. Activation of the LCipsi or LCipsi/contra→SC dampened pain-like behavior. Moreover, Designer Receptor Exclusively Activated by Designer Drugs (DREADDs)-mediated inactivation of the CCI-ST LCipsi→rACC or CCI-LT LCipsi/contra→rACC pathway, or intra-rACC antagonism of α-adrenoreceptors, also dampens pain-like behavior. CONCLUSIONS: In the short term, activation of the LC after CCI attenuates spontaneous pain-like behaviors via projections to the SC while increasing nociception via projections to the rACC. In the long term, only the projections from the LC to the rACC contribute to modulate pain-like behaviors in this model.

Crosstalk between P2Y receptors and cyclooxygenase activity in inflammation and tissue repair.

The role of extracellular nucleotides as modulators of inflammation and cell stress is well established. One of the main actions of these molecules is mediated by the activation of purinergic receptors (P2) of the plasma membrane. P2 receptors can be classified according to two different structural families: P2X ionotropic ion channel receptors, and P2Y metabotropic G protein-coupled receptors. During inflammation, damaged cells release nucleotides and purinergic signaling occurs along the temporal pattern of the synthesis of pro-inflammatory and pro-resolving mediators by myeloid and lymphoid cells. In macrophages under pro-inflammatory conditions, the expression and activity of cyclooxygenase 2 significantly increases and enhances the circulating levels of prostaglandin E2 (PGE2), which exerts its effects both through specific plasma membrane receptors (EP1-EP4) and by activation of intracellular targets. Here we review the mechanisms involved in the crosstalk between PGE2 and P2Y receptors on macrophages, which is dependent on several isoforms of protein kinase C and protein kinase D1. Due to this crosstalk, a P2Y-dependent increase in calcium is blunted by PGE2 whereas, under these conditions, macrophages exhibit reduced migratory capacity along with enhanced phagocytosis, which contributes to the modulation of the inflammatory response and tissue repair.

Lipoxin-mediated signaling: ALX/FPR2 interaction and beyond.

In the aftermath of tissue injury or infection, an efficient resolution mechanism is crucial to allow tissue healing and preserve appropriate organ functioning. Pro-resolving bioactive lipids prevent uncontrolled inflammation and its consequences. Among these mediators, lipoxins were the first described and their pro-resolving actions have been mainly described in immune cells. They exert their actions mostly through formyl-peptide receptor 2 (ALX/FPR2 receptor), a G-protein-coupled receptor whose biological function is tremendously complex, primarily due to its capacity to mediate variable cellular responses. Moreover, lipoxins can also interact with alternative receptors like the cytoplasmic aryl hydrocarbon receptor, the cysteinyl-leukotrienes receptors or GPR32, triggering different intracellular signaling pathways. The available information about this complex response mediated by lipoxins is addressed in this review, going over the different mechanisms used by these molecules to stop the inflammatory reaction and avoid the development of dysregulated and chronic pathologies.

Impact of supplementation with dihydroxylated vitamin D3 on performance parameters and gut health in weaned Iberian piglets under indoor/outdoor conditions.

BACKGROUND: Vitamin D may improve innate antimicrobial response and the integrity of the intestinal mucosal barrier representing an alternative to antibiotics for improving pig health. Therefore, benefits of dietary supplementation with a product based on vitamin D3 metabolite-rich plant extracts were assessed in 252 purebred Iberian piglets for a period of 60 days. The study group received 1,25 dihydroxyvitamin D (1,25(OH)2D) (100 ppm) in the conventional feed, which already included vitamin D (2000 IU in the starter and 1000 IU in the adaptation diets, respectively). Average daily gain (ADG), feed conversion ratio (FCR) and coefficient of variation of body weight (CV-BW) were assessed along the study. Blood samples, from 18 animals of the study group and 14 animals of the control group, were collected at selected time points to determine white blood cell count, concentration of vitamin D3 and its metabolites, and IgA and IgG in serum. Histopathology, morphometry, and immunohistochemistry (IgA and FoxP3) from small intestine samples were performed on days 30 and 60 of the study from 3 animals per group and time point. RESULTS: The ADG (493 vs 444 g/day) and FCR (2.3 vs 3.02) showed an improved performance in the supplemented animals. Moreover, the lower CV-BW indicated a greater homogeneity in the treated batches (13.17 vs 26.23%). Furthermore, a mild increase of IgA and in the number of regulatory T cells in the small intestine were observed in treated pigs. CONCLUSIONS: These results highlight the benefits of this supplementation and encourage to develop further studies along other production stages.

Beneficial effect of TLR4 blockade by a specific aptamer antagonist after acute myocardial infarction.

Experimental evidence indicates that the control of the inflammatory response after myocardial infarction is a key strategy to reduce cardiac injury. Cellular damage after blood flow restoration in the heart promotes sterile inflammation through the release of molecules that activate pattern recognition receptors, among which TLR4 is the most prominent. Transient regulation of TLR4 activity has been considered one of the potential therapeutic interventions with greater projection towards the clinic. In this regard, the characterization of an aptamer (4FT) that acts as a selective antagonist for human TLR4 has been investigated in isolated macrophages from different species and in a rat model of cardiac ischemia/reperfusion (I/R). The binding kinetics and biological responses of murine and human macrophages treated with 4FT show great affinity and significant inhibition of TLR4 signaling including the NF-κB pathway and the LPS-dependent increase in the plasma membrane currents (Kv currents). In the rat model of I/R, administration of 4FT following reoxygenation shows amelioration of cardiac injury function and markers, a process that is significantly enhanced when the second dose of 4FT is administered 24 h after reperfusion of the heart. Parameters such as cardiac injury biomarkers, infiltration of circulating inflammatory cells, and the expression of genes associated with the inflammatory onset are significantly reduced. In addition, the expression of anti-inflammatory genes, such as IL-10, and pro-resolution molecules, such as resolvin D1 are enhanced after 4FT administration. These results indicate that targeting TLR4 with 4FT offers new therapeutic opportunities to prevent cardiac dysfunction after infarction.

Combined Immune Defect in B-Cell Lymphoproliferative Disorders Is Associated with Severe Infection and Cancer Progression.

B cell chronic lymphoproliferative diseases (B-CLPD) are associated with secondary antibody deficiency and other innate and adaptive immune defects, whose impact on infectious risk has not been systematically addressed. We performed an immunological analysis of a cohort of 83 B-CLPD patients with recurrent and/or severe infections to ascertain the clinical relevance of the immune deficiency expression. B-cell defects were present in all patients. Patients with combined immune defect had a 3.69-fold higher risk for severe infection (p = 0.001) than those with predominantly antibody defect. Interestingly, by Kaplan-Meier analysis, combined immune defect showed an earlier progression of cancer with a hazard ratio of 3.21, than predominantly antibody defect (p = 0.005). When B-CLPD were classified in low-degree, high-degree, and plasma cell dyscrasias, risk of severe disease and cancer progression significantly diverged in combined immune defect, compared with predominantly antibody defect (p = 0.001). Remarkably, an underlying primary immunodeficiency (PID) was suspected in 12 patients (14%), due to prior history of infections, autoimmune and granulomatous conditions, atypical or variegated course and compatible biological data. This first proposed SID classification might have relevant clinical implications, in terms of predicting severe infections and cancer progression, and might be applied to different B-CLPD entities.

Specialized Proresolving Mediators Protect Against Experimental Autoimmune Myocarditis by Modulating Ca2+ Handling and NRF2 Activation.

Specialized proresolving mediators and, in particular, 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester (BML-111) emerge as new therapeutic tools to prevent cardiac dysfunction and deleterious cardiac damage associated with myocarditis progression. The cardioprotective role of BML-111 is mainly caused by the prevention of increased oxidative stress and nuclear factor erythroid-derived 2-like 2 (NRF2) down-regulation induced by myocarditis. At the molecular level, BML-111 activates NRF2 signaling, which prevents sarcoplasmic reticulum-adenosine triphosphatase 2A down-regulation and Ca2+ mishandling, and attenuates the cardiac dysfunction and tissue damage induced by myocarditis.

High-fat diet activates splenic NOD1 and enhances neutrophil recruitment and neutrophil extracellular traps release in the spleen of ApoE-deficient mice.

In the course of atherogenesis, the spleen plays an important role in the regulation of extramedullary hematopoiesis, and in the control of circulating immune cells, which contributes to plaque progression. Here, we have investigated the role of splenic nucleotide-binding oligomerization domain 1 (NOD1) in the recruitment of circulating immune cells, as well as the involvement of this immune organ in extramedullary hematopoiesis in mice fed on a high-fat high-cholesterol diet (HFD). Under HFD conditions, the absence of NOD1 enhances the mobilization of immune cells, mainly neutrophils, from the bone marrow to the blood. To determine the effect of NOD1-dependent mobilization of immune cells under pro-atherogenic conditions, Apoe-/- and Apoe-/-Nod1-/- mice fed on HFD for 4 weeks were used. Splenic NOD1 from Apoe-/- mice was activated after feeding HFD as inferred by the phosphorylation of the NOD1 downstream targets RIPK2 and TAK1. Moreover, this activation was accompanied by the release of neutrophil extracellular traps (NETs), as determined by the increase in the expression of peptidyl arginine deiminase 4, and the identification of citrullinated histone H3 in this organ. This formation of NETs was significantly reduced in Apoe-/-Nod1-/- mice. Indeed, the presence of Ly6G+ cells and the lipidic content in the spleen of mice deficient in Apoe and Nod1 was reduced when compared to the Apoe-/- counterparts, which suggests that the mobilization and activation of circulating immune cells are altered in the absence of NOD1. Furthermore, confirming previous studies, Apoe-/-Nod1-/- mice showed a reduced atherogenic disease, and diminished recruitment of neutrophils in the spleen, compared to Apoe-/- mice. However, splenic artery ligation reduced the atherogenic burden in Apoe-/- mice an effect that, unexpectedly was lost in Apoe-/-Nod1-/- mice. Together, these results suggest that neutrophil accumulation and activity in the spleen are driven in part by NOD1 activation in mice fed on HFD, contributing in this way to regulating atherogenic progression.

The Aryl Hydrocarbon Receptor Ligand FICZ Improves Left Ventricular Remodeling and Cardiac Function at the Onset of Pressure Overload-Induced Heart Failure in Mice.

Adverse ventricular remodeling is the heart's response to damaging stimuli and is linked to heart failure and poor prognosis. Formyl-indolo [3,2-b] carbazole (FICZ) is an endogenous ligand for the aryl hydrocarbon receptor (AhR), through which it exerts pleiotropic effects including protection against inflammation, fibrosis, and oxidative stress. We evaluated the effect of AhR activation by FICZ on the adverse ventricular remodeling that occurs in the early phase of pressure overload in the murine heart induced by transverse aortic constriction (TAC). Cardiac structure and function were evaluated by cardiac magnetic resonance imaging (CMRI) before and 3 days after Sham or TAC surgery in mice treated with FICZ or with vehicle, and cardiac tissue was used for biochemical studies. CMRI analysis revealed that FICZ improved cardiac function and attenuated cardiac hypertrophy. These beneficial effects involved the inhibition of the hypertrophic calcineurin/NFAT pathway, transcriptional reduction in pro-fibrotic genes, and antioxidant effects mediated by the NRF2/NQO1 pathway. Overall, our findings provide new insight into the role of cardiac AhR signaling in the injured heart.

Downlink Performance Modeling and Evaluation of Batteryless Low Power BLE Node.

Deploying low maintenance and long-life systems is an important requirement of emerging commercial Internet of Things (IoT) solutions. Such systems can be envisioned in which the connected devices are powered by energy harvested from ambient sources and stored in long-lifetime capacitors rather than short-lived and polluting batteries. However, due to the unpredictable nature of ambient energy harvesting, such batteryless IoT devices might not always have enough energy to initiate communication. The Bluetooth Low Energy (BLE) specification defines support for Low Power Nodes (LPNs) using the friendship feature, where the LPN is associated with a neighbouring friend node (FN). The LPN can receive downlink (DL) data and remain connected to the network via the FN that buffers the LPN's incoming packets while allowing the LPN to save energy by sleeping or turning itself off. This novel BLE feature makes the LPN highly suitable to support the connection of batteryless ambiently-powered IoT devices. While the LPN can decide when to transmit uplink (UL) packets and does not depend on the FN to receive downlink (DL) data, the LPN needs to poll its FN to receive the buffered packets. However, the DL packet latency increases with this process due to the buffering time at the FN. Therefore, in this work, we present an analytical model to characterize the performance as a function of DL data latency and packet delivery ratio (PDR) of a batteryless LPN powered by different harvesting powers and capacitor sizes. This would help to optimally choose the correct configuration of the batteryless LPN for its network deployment. We also compare the analytical model and simulation results, showing consistency with an average error of 2.23% for DL data latency and 0.09% for the PDR.

NOD1 splenic activation confers ferroptosis protection and reduces macrophage recruitment under pro-atherogenic conditions.

The bioavailability and regulation of iron is essential for central biological functions in mammals. The role of this element in ferroptosis and the dysregulation of its metabolism contribute to diseases, ranging from anemia to infections, alterations in the immune system, inflammation and atherosclerosis. In this sense, monocytes and macrophages modulate iron metabolism and splenic function, while at the same time they can worsen the atherosclerotic process in pathological conditions. Since the nucleotide-binding oligomerization domain 1 (NOD1) has been linked to numerous disorders, including inflammatory and cardiovascular diseases, we investigated its role in iron homeostasis. The iron content was measured in various tissues of Apoe-/- and Apoe-/-Nod1-/- mice fed a high-fat diet (HFD) for 4 weeks, under normal or reduced splenic function after ligation of the splenic artery. In the absence of NOD1 the iron levels decreased in spleen, heart and liver regardless the splenic function. This iron decrease was accompanied by an increase in the recruitment of F4/80+-macrophages in the spleen through a CXCR2-dependent signaling, as deduced by the reduced recruitment after administration of a CXCR2 inhibitor. CXCR2 mediates monocyte/macrophage chemotaxis to areas of inflammation and accumulation of leukocytes in the atherosclerotic plaque. Moreover, in the absence of NOD1, inhibition of CXCR2 enhanced atheroma progression. NOD1 activation increased the levels of GPX4 and other iron and ferroptosis regulatory proteins in macrophages. Our findings highlight the preeminent role of NOD1 in iron homeostasis and ferroptosis. These results suggest promising avenues of investigation for the diagnosis and treatment of iron-related diseases directed by NOD1.

The Role of the Locus Coeruleus in Pain and Associated Stress-Related Disorders.

The locus coeruleus (LC)-noradrenergic system is the main source of noradrenaline in the central nervous system and is involved intensively in modulating pain and stress-related disorders (e.g., major depressive disorder and anxiety) and in their comorbidity. However, the mechanisms involving the LC that underlie these effects have not been fully elucidated, in part owing to the technical difficulties inherent in exploring such a tiny nucleus. However, novel research tools are now available that have helped redefine the LC system, moving away from the traditional view of LC as a homogeneous structure that exerts a uniform influence on neural activity. Indeed, innovative techniques such as DREADDs (designer receptors exclusively activated by designer drugs) and optogenetics have demonstrated the functional heterogeneity of LC, and novel magnetic resonance imaging applications combined with pupillometry have opened the way to evaluate LC activity in vivo. This review aims to bring together the data available on the efferent activity of the LC-noradrenergic system in relation to pain and its comorbidity with anxiodepressive disorders. Acute pain triggers a robust LC stress response, producing spinal cord-mediated endogenous analgesia while promoting aversion, vigilance, and threat detection through its ascending efferents. However, this protective biological system fails in chronic pain, and LC activity produces pain facilitation, anxiety, increased aversive memory, and behavioral despair, acting at the medulla, prefrontal cortex, and amygdala levels. Thus, the activation/deactivation of specific LC projections contributes to different behavioral outcomes in the shift from acute to chronic pain.

The anti-aging factor Klotho protects against acquired long QT syndrome induced by uremia and promoted by fibroblast growth factor 23.

BACKGROUND: Chronic kidney disease (CKD) is associated with increased propensity for arrhythmias. In this context, ventricular repolarization alterations have been shown to predispose to fatal arrhythmias and sudden cardiac death. Between mineral bone disturbances in CKD patients, increased fibroblast growth factor (FGF) 23 and decreased Klotho are emerging as important effectors of cardiovascular disease. However, the relationship between imbalanced FGF23-Klotho axis and the development of cardiac arrhythmias in CKD remains unknown. METHODS: We carried out a translational approach to study the relationship between the FGF23-Klotho signaling axis and acquired long QT syndrome in CKD-associated uremia. FGF23 levels and cardiac repolarization dynamics were analyzed in patients with dialysis-dependent CKD and in uremic mouse models of 5/6 nephrectomy (Nfx) and Klotho deficiency (hypomorphism), which show very high systemic FGF23 levels. RESULTS: Patients in the top quartile of FGF23 levels had a higher occurrence of very long QT intervals (> 490 ms) than peers in the lowest quartile. Experimentally, FGF23 induced QT prolongation in healthy mice. Similarly, alterations in cardiac repolarization and QT prolongation were observed in Nfx mice and in Klotho hypomorphic mice. QT prolongation in Nfx mice was explained by a significant decrease in the fast transient outward potassium (K+) current (Itof), caused by the downregulation of K+ channel 4.2 subunit (Kv4.2) expression. Kv4.2 expression was also significantly reduced in ventricular cardiomyocytes exposed to FGF23. Enhancing Klotho availability prevented both long QT prolongation and reduced Itof current. Likewise, administration of recombinant Klotho blocked the downregulation of Kv4.2 expression in Nfx mice and in FGF23-exposed cardiomyocytes. CONCLUSION: The FGF23-Klotho axis emerges as a new therapeutic target to prevent acquired long QT syndrome in uremia by minimizing the predisposition to potentially fatal ventricular arrhythmias and sudden cardiac death in patients with CKD.

Nerve injury induces transient locus coeruleus activation over time: role of the locus coeruleus-dorsal reticular nucleus pathway.

ABSTRACT: The transition from acute to chronic pain results in maladaptive brain remodeling, as characterized by sensorial hypersensitivity and the ensuing appearance of emotional disorders. Using the chronic constriction injury of the sciatic nerve as a model of neuropathic pain in male Sprague-Dawley rats, we identified time-dependent plasticity of locus coeruleus (LC) neurons related to the site of injury, ipsilateral (LCipsi) or contralateral (LCcontra) to the lesion, hypothesizing that the LC→dorsal reticular nucleus (DRt) pathway is involved in the pathological nociception associated with chronic pain. LCipsi inactivation with lidocaine increased cold allodynia 2 days after nerve injury but not later. However, similar blockade of LCcontra reduced cold allodynia 7 and 30 days after inducing neuropathy but not earlier. Furthermore, lidocaine blockade of the LCipsi or LCcontra reversed pain-induced depression 30 days after neuropathy. Long-term pain enhances phosphorylated cAMP-response element binding protein expression in the DRtcontra but not in the DRtipsi. Moreover, inactivation of the LCcontra→DRtcontra pathway using dual viral-mediated gene transfer of designer receptor exclusively activated by designer drugs produced consistent analgesia in evoked and spontaneous pain 30 days postinjury. This analgesia was similar to that produced by spinal activation of α2-adrenoreceptors. Furthermore, chemogenetic inactivation of the LCcontra→DRtcontra pathway induced depressive-like behaviour in naïve animals, but it did not modify long-term pain-induced depression. Overall, nerve damage activates the LCipsi, which temporally dampens the neuropathic phenotype. However, the ensuing activation of a LCcontra→DRtcontra facilitatory pain projection contributes to chronic pain, whereas global bilateral LC activation contributes to associated depressive-like phenotype.

Pain and depression comorbidity causes asymmetric plasticity in the locus coeruleus neurons.

There is strong comorbidity between chronic pain and depression, although the neural circuits and mechanisms underlying this association remain unclear. By combining immunohistochemistry, tracing studies and western blotting, with the use of different DREADDS (designer receptor exclusively activated by designer drugs) and behavioural approaches in a rat model of neuropathic pain (chronic constriction injury), we explore how this comorbidity arises. To this end, we evaluated the time-dependent plasticity of noradrenergic locus coeruleus neurons relative to the site of injury: ipsilateral (LCipsi) or contralateral (LCcontra) locus coeruleus at three different time points: short (2 days), mid (7 days) and long term (30-35 days from nerve injury). Nerve injury led to sensorial hypersensitivity from the onset of injury, whereas depressive-like behaviour was only evident following long-term pain. Global chemogenetic blockade of the LCipsi system alone increased short-term pain sensitivity while the blockade of the LCipsi or LCcontra relieved pain-induced depression. The asymmetric contribution of locus coeruleus modules was also evident as neuropathy develops. Hence, chemogenetic blockade of the LCipsi→spinal cord projection, increased pain-related behaviours in the short term. However, this lateralized circuit is not universal as the bilateral chemogenetic inactivation of the locus coeruleus-rostral anterior cingulate cortex pathway or the intra-rostral anterior cingulate cortex antagonism of alpha1- and alpha2-adrenoreceptors reversed long-term pain-induced depression. Furthermore, chemogenetic locus coeruleus to spinal cord activation, mainly through LCipsi, reduced sensorial hypersensitivity irrespective of the time post-injury. Our results indicate that asymmetric activation of specific locus coeruleus modules promotes early restorative analgesia, as well as late depressive-like behaviour in chronic pain and depression comorbidity.

The Informal Social Support for Autonomy and Dependence in Pain Inventory Spanish version.

Social support plays a crucial role in the quality of life of people with chronic pain. The Informal Social Support for Autonomy and Dependence in Pain Inventory assesses two functions of received social support: the promotion of autonomy and the promotion of dependence. The aim of this cross-sectional study was to adapt this instrument for its use in the Spanish population. The sample comprised 256 individuals with chronic pain. Participants were recruited through two local associations of people with fibromyalgia, a physiotherapy unit and a hospital pain unit. The data were collected in Spain between October 2018 and January 2020. The structure of the questionnaire was analysed using confirmatory factor analysis, average variance extracted, composite reliability and internal consistency indexes, and inter-correlations between the scales. The criterion-related validity of the instrument was analysed by investigating its relationship with pain intensity, positive and negative affect, daily functioning, activity impairment, wellbeing and satisfaction with life. The structure with the best fit had four related factors: emotional social support for the promotion of autonomy; instrumental social support for the promotion of autonomy; emotional social support for the promotion of dependence and instrumental social support for the promotion of dependence. The scales showed adequate internal consistency. An association was found between higher levels of instrumental social support for the promotion of dependence and higher levels of pain-related disability and decreased daily functioning. An association was also found between the promotion of autonomy and increased satisfaction with life. The Spanish version of the inventory shows appropriate psychometric properties. In the setting of disability prevention, this instrument is useful in assessing the support relationships between people with chronic pain and their relatives.