A multifunctional microRNA, miR-155, has been recently recognized as an important modulator of numerous biological processes. In our previous in vitro studies, miR-155 was identified as a potential regulator of the endothelial morphogenesis. The present study demonstrates that in vivo inhibition of miR-155 supports cerebral vasculature after experimental stroke. Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Microvasculature in peri-infarct area, infarct size, and animal functional recovery were assessed at 1, 2, and 3 weeks after dMCAO. Using in vivo two-photon microscopy, we detected improved blood flow and microvascular integrity in the peri-infarct area of miR-155 inhibitor-injected mice. Electron microscopy revealed that, in contrast to the control group, these animals demonstrated well preserved capillary tight junctions (TJs). Western blot analysis data indicate that improved TJ integrity in the inhibitor-injected animals could be associated with stabilization of the TJ protein ZO-1 and mediated by the miR-155 target protein Rheb. MRI analysis showed significant (34%) reduction of infarct size in miR-155 inhibitor-injected animals at 21 d after dMCAO. Reduced brain injury was confirmed by electron microscopy demonstrating decreased neuronal damage in the peri-infarct area of stroke. Preservation of brain tissue was reflected in efficient functional recovery of inhibitor-injected animals. Based on our findings, we propose that in vivo miR-155 inhibition after ischemia supports brain microvasculature, reduces brain tissue damage, and improves the animal functional recovery.

Disturbances of circadian rhythms have been associated with obesity and type 2 diabetes. The nuclear receptor Rev-erbα was suggested to link circadian rhythms and metabolism in peripheral tissues. The aim of the present study was to dissect the role of this clock gene in the pancreatic β-cell function and to analyze whether its expression is modulated by leptin and diet-induced obesity. To address the function of Rev-erbα, we used small interfering RNA in mouse islet cells and in MIN-6 cells. Cell proliferation was measured by bromodeoxyuridine incorporation, apoptosis by the terminal deoxynucleotidyl transferase dUTP nick end labeling technique, insulin secretion by RIA, and gene expression by RT-PCR. Pancreatic islets were isolated at different zeitgeber times 0, 6, and 12 after 6 wk of high-fat diet treatment, and then gene expression and insulin secretion were determined. Rev-erbα down-regulation by small interfering RNA treatment in islet cells and MIN-6 cells impaired glucose-induced insulin secretion, decreased the expression of key lipogenic genes, and inhibited β-cell proliferation. In vivo and in vitro leptin treatment increased Rev-erbα expression in isolated islets through a MAPK pathway. High-fat diet treatment disrupted the circadian Rev-erbα gene expression profile along with insulin secretion, indicating an important role of this clock gene in β-cell function. These results indicate that the clock gene Rev-erbα plays multiple functions in the pancreatic β-cell. Although the increase in Rev-erbα expression may promote β-cell adaptation in different metabolic situations, its deregulation may lead to altered β-cell function.

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene that codes for the chloride channel, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CF patients are frequently colonized by several pathogens, but the mechanisms that allow colonization in spite of apparently functional immune systems are incompletely understood. In this paper we show that blood peripheral monocytes isolated from CF patients are found in an endotoxin tolerance state, yet this is not due to a deficient TLR activation. On the other hand, levels of the amplifier of inflammatory responses, TREM-1 (Triggering Receptor Expressed on Myeloid cells), are notably down-regulated in monocytes from patients, in comparison to those extracted from healthy volunteers. Furthermore, the soluble form of TREM-1 (sTREM-1) was not detected in the sera of patients. Additionally, and in strict contrast to patients who suffer from Chronic Obstructive Pulmonary Disease (COPD), CF monocytes challenged ex vivo with LPS neither up-regulated membrane-anchored TREM-1 nor sTREM-1. Finally, similar levels of PGE2 expression and p65 translocation into the nucleus were found in both patients and healthy volunteers, thus suggesting that TREM-1 regulation is neither controlled by PGE2 levels nor by p65 activation in this case. However, PU.1 translocation into the nucleus was significantly higher in CF monocytes than in controls, suggesting a role for this transcription factor in the control of TREM-1 expression. We conclude that down-regulation of TREM-1 expression in cystic fibrosis patients is at least partly responsible for the endotoxin tolerance state in which their monocytes are locked.

Leptin plays an important role in the control of food intake, energy expenditure, metabolism, and body weight. This hormone also has a key function in the regulation of glucose homeostasis. Although leptin acts through central and peripheral mechanisms to modulate glucose metabolism, the pancreatic b-cell of the endocrine pancreas is a critical target of leptin actions. Leptin receptors are present in the b-cell, and their activation directly inhibits insulin secretion from these endocrine cells. The effects of leptin on insulin occur also in the long term, since this hormone inhibits insulin gene expression as well. Additionally, b-cell mass can be affected by leptin through changes in proliferation, apoptosis, or cell size. All these different functions in the b-cell are triggered by leptin as a result of the large diversity of signaling pathways that this hormone is able to activate in the endocrine pancreas. Therefore, leptin can participate in glucose homeostasis owing to different levels of modulation of the pancreatic b-cell population. Furthermore, it has been proposed that alterations in this level of regulation could contribute to the impairment of b-cell function in obesity states. In the present review, we will discuss all these issues with special emphasis on the effects and pathways of leptin signaling in the pancreatic b-cell.

Insulin resistance and hyperinsulinemia are generally associated with obesity. Obese nondiabetic individuals develop a compensatory β-cell response to adjust insulin levels to the increased demand, maintaining euglycemia. Although several studies indicate that this compensation relies on structural changes, the existence of β-cell functional adaptations is incompletely understood. Here, we fed female mice with a high-fat diet (HFD) for 12 weeks. These animals became obese, hyperinsulinemic, insulin-resistant, and mildly glucose-intolerant while fed, and fasting glycemia was comparable in HFD and control mice. Islets from HFD animals exhibited increased β-cell mass and hypertrophy. Additionally, they had enhanced insulin gene expression and content and augmented glucose-induced insulin secretion. Electrophysiological examination of β-cells from both groups showed no differences in KATP channel open probability and conductance. However, action potentials elicited by glucose had larger amplitude in obese mice. Glucose-induced Ca²⁺ signals in intact islets, in isolated β-cells, and individual β-cells within islets were also increased in HFD mice. Additionally, a higher proportion of glucose-responsive cells was present in obese mice. In contrast, whole-cell Ca²⁺ current densities were similar in both groups. Capacitance measurements showed that depolarization-evoked exocytosis was enhanced in HFD β-cells compared with controls. Although this augment was not significant when capacitance increases of the whole β-cell population were normalized to cell size, the exocytotic output varied significantly when β-cells were distributed by size ranges. All these findings indicate that β-cell functional adaptations are present in the islet compensatory response to obesity.

BackgroundMicroRNA miR-155 is implicated in modulation of the inflammatory processes in various pathological conditions. In our previous studies, we demonstrated that in vivo inhibition of miR-155 promotes functional recovery after mouse experimental stroke. In the present study, we explored if this beneficial effect is associated with miR-155 inhibition-induced alterations in post-stroke inflammatory response.MethodsIntravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Temporal changes in the expression of cytokines and key molecules associated with cytokine signaling were assessed at 7, 14, and 21 days after dMCAO, using mouse cytokine gene and protein arrays and Western blot analyses. Electron and immunofluorescence confocal microscopy techniques were used to evaluate the ultrastructural changes, as well as altered expression of specific phenotypic markers, at different time points after dMCAO.ResultsIn the inhibitor-injected mice (inhibitor group), there was a significant decrease in CCL12 and CXCL3 cytokine expression at 7 days and significantly increased levels of major cytokines IL-10, IL-4, IL-6, MIP-1α, IL-5, and IL-17 at 14 days after dMCAO. These temporal changes correlated with altered expression of miR-155 target proteins SOCS-1, SHIP-1, and C/EBP-β and phosphorylation levels of cytokine signaling regulator STAT-3. Electron microscopy showed decreased number of phagocytically active peri-vascular microglia/macrophages in the inhibitor samples. Immunofluorescence and Western blot of these samples demonstrated that expression of leukocyte/ macrophage marker CD45 and phagocytosis marker CD68 was reduced at 7 days, and in contrast, significantly increased at 14 days after dMCAO, as compared to controls.ConclusionsBased on our findings, we propose that in vivo miR-155 inhibition following mouse stroke significantly alters the time course of the expression of major cytokines and inflammation-associated molecules, which could influence inflammation process and tissue repair after experimental cerebral ischemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0753-x) contains supplementary material, which is available to authorized users.

How stable and general is behavior once reached maximum learning? To answer this question and understand post-acquisition behavior and its related individual differences, we propose a psychological principle that naturally extends the basic associative single-cue Rescorla-Wagner model (which may also be called Hull model) of Pavlovian conditioning to (i) a framework of dynamical models predicting resistance to learning in the first few sessions followed by an over-optimal response peak. In turn, (ii) the theory can be further extended to describe response fluctuations by the laws of quantum mechanics. (iii) We also introduce an independent model characterized by the presence of a stochastic noise of cognitive origin. We ran an unusually long experiment with 32 rats over 3960 trials, where we excluded habituation and other well-known phenomena as sources of variability in the subjects' performance. There is weak to positive evidence that Hull model is the best nonlinear regression to averaged data only for a minority of the subjects, while its dynamical extension (i) can explain the totality of data. The noise encountered in all individual responses is white, thus confirming the simplest version of model (iii) but standing in contrast with the colored-noise findings in human performance. Finally, data are compatible with the quantum extension (ii), although experimental uncertainties dominate the result. On the other hand, data do not favor models with (iv) a long-memory effect or (v) where response variability is solely described by a random fractal.

Evaluation of functional outcome is widely used across species to assess the recovery process following various pathological conditions, including spinal cord injury, musculo-skeletal injury, mithochondrial disease, neuropathic cancer, Huntington's disease, chronic pain, cortical lesion, and olivocerebellar degeneration among others. The Stroke Therapy Academic Industry Roundtable (STAIR) recommends multiple endpoints for behavioral studies in pre-clinical stroke research, to demonstrate their clinical relevance. One of the more challenging tasks in experimental stroke research is measuring long-term functional outcome in mice. It is, however, becoming more important, since transgenic mice are increasingly used for modeling human neurological disorders. Using CatWalk, we characterized long-lasting gait/locomotion deficits following mouse distal middle cerebral artery occlusion (dMCAO). The post-dMCAO assessment was performed at 7, 14, 21, and 28 days after experimental ischemia. When compared to sham-operated mice, dMCAO animals displayed a statistically significant decrease in Spatial parameters (such as Paw Area), while the Temporal parameters (Stand, Initial and Terminal Dual Stances) were significantly increased for three weeks after surgery. Kinetic parameters were significantly decreased in dMCAO animals at 7 days after dMCAO. The Interlimb coordination group of parameters displayed the strongest deficits at 21 days. While CatWalk variables were altered in all paws, the degree of change was greatest for the parameters measured from the Right Front Paw (contralateral to the lesion). All parameters measured in dMCAO and Sham-operated groups reached similar levels at four weeks after the experimental insult, which reflects a spontaneous post-ischemic recovery. Based on our investigation, we conclude that CatWalk represents a relevant and sensitive analysis, which allows long-term characterization of animal functional recovery in the dMCAO model of experimental ischemia.