Effect of NF kappa B Inhibition by CAPE on Skeletal Muscle Ischemia-Reperfusion Injury

Background/Aims. Nuclear factor kappa B (NF kappa B) plays important role in the pathogenesis of skeletal muscle ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent NF kappa B inhibitor, exhibits protective effects on I/R injury in some tissues. In this report, the effect of CAPE on skeletal muscle I/R injury in rats was studied. Methods. Wistar rats were submitted to sham operation, 120-min hindlimb ischemia, or 120-min hindlimb ischemia plus saline or CAPE treatment followed by 4-h reperfusion. Gastrocnemius muscle injury was evaluated by serum aminotransferase levels, muscle edema, tissue glutathione and malondialdehyde measurement, and scoring of histological damage. Apoptotic nuclei were determined by a terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay. Muscle neutrophil and mast cell accumulation were also assessed. Lipoperoxidation products and NF kappa B were evaluated by 4-hydroxynonenal and NF kappa B p65 immunohistochemistry, respectively. Results. Animals submitted to ischemia showed a marked increase in aminotransferases after reperfusion, but with lower levels in the CAPE group. Tissue glutathione levels declined gradually during ischemia to reperfusion, and were partially recovered with CAPE treatment. The histological damage score, muscle edema percentage, tissue malondialdehyde content, apoptosis index, and neutrophil and mast cell infiltration, as well as 4-hydroxynonenal and NF kappa B p65 labeling, were higher in animals submitted to I/R compared with the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. Conclusions. CAPE was able to protect skeletal muscle against I/R, injury in rats. This effect may be associated with the inhibition of the NF kappa B signaling pathway and decrease of the tissue inflammatory response following skeletal muscle I/R. (C) 2009 Elsevier Inc. All rights reserved.

Consequences of lifetime isolated growth hormone (GH) deficiency and effects of short-term GH treatment on bone in adults with a mutation in the GHRH-receptor gene

Growth hormone (GH) influences bone mass maintenance. However, the consequences of lifetime isolated GH deficiency (IGHD) on bone are not well established. We assessed the bone status and the effect of 6 months of GH replacement in GH-naive adults with IGHD due to a homozygous mutation of the GH-releasing hormone (GHRH)-receptor gene (GHRHR). We studied 20 individuals (10 men) with IGHD at baseline, after 6 months of depot GH treatment, and 6 and 12 months after discontinuation of GH. Quantitative ultrasound (QUS) of the heel was performed and serum osteocalcin (OC) and C-terminal cross-linking telopeptide of type I collagen (ICTP) were measured. QUS was also performed at baseline and 12 months later in a group of 20 normal control individuals (CO), who did not receive GH treatment. At baseline, the IGHD group had a lower T-score on QUS than CO (-1.15 +/- 0.9 vs. -0.07 +/- 0.9, P < 0.001). GH treatment improved this parameter, with improvement persisting for 12 months post-treatment (T-score for IGHD = -0.59 +/- 0.9, P < 0.05). GH also caused an increase in serum OC (baseline vs. pGH, P < 0.001) and ICTP (baseline vs. pGH, P < 0.01). The increase in OC was more marked during treatment and its reduction was slower after GH discontinuation than in ICTP. These data suggest that lifetime severe IGHD is associated with significant reduction in QUS parameters, which are partially reversed by short-term depot GH treatment. The treatment induces a biochemical pattern of bone anabolism that persists for at least 6 months after treatment discontinuation.

Improved Endothelial Function and Reversal of Myocardial Perfusion Defects after Aerobic Physical Training in a Patient with Microvascular Myocardial Ischemia

The objective of this report is to document the effects of an aerobic training program on myocardial perfusion, and endothelial function abnormalities, and on the relief of angina in a patient with microvascular myocardial ischemia. A 53-year-old female patient exhibited precordial pain on effort and angiographically normal coronaries. Her symptoms had been present for 4 yrs despite pharmacologic treatment for the control of risk factors, with myocardial perfusion scintigraphy revealing an extensive reversible perfusion defect. She was submitted to aerobic training for 4 mos, obtaining significant improvement of the anginal symptoms. Additionally, after the aerobic training program, scintigraphy revealed the disappearance of the myocardial perfusion defect, with a marked improvement of endothelium-dependent vasodilatory response and an improved quality-of-life score. These results suggest that aerobic training can improve endothelial function, leading to a reduction of ischemia and an improved quality-of-life in patients with microvascular myocardial ischemia.

IL-17 Receptor Signaling Is Required to Control Polymicrobial Sepsis

Sepsis is a systemic inflammatory response resulting from the inability of the host to contain the infection locally. Previously, we demonstrated that during severe sepsis there is a marked failure of neutrophil migration to the infection site, which contributes to dissemination of infection, resulting in high mortality. IL-17 plays an important role in neutrophil recruitment. Herein, we investigated the role of IL-17R signaling in polymicrobial sepsis induced by cecal ligation and puncture (CLP). It was observed that IL-17R-deficient mice, subjected to CLP-induced non-severe sepsis, show reduced neutrophil recruitment into the peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared with C57BL/6 littermates. As a consequence, the mice showed an increased mortality rate. The ability of IL-17 to induce neutrophil migration was demonstrated in vivo and in vitro. Beside its role in neutrophil recruitment to the infection focus, IL-17 enhanced the microbicidal activity of the migrating neutrophils by a mechanism dependent on NO. Therefore, IL-17 plays a critical role in host protection during polymicrobial sepsis. The Journal of Immunology, 2009, 182: 7846-7854.

Characterization and pharmacological evaluation of febrile response on zymosan-induced arthritis in rats

Kanashiro A, Pessini AC, Machado RR, Malvar DC, Aguiar FA, Soares DM, Vale ML, Souza GEP. Characterization and pharmacological evaluation of febrile response on zymosan-induced arthritis in rats. Am J Physiol Regul Integr Comp Physiol 296: R1631-R1640, 2009. First published February 25, 2009; doi:10.1152/ajpregu.90527.2008.-The present study investigated the febrile response in zymosan-induced arthritis, as well as the increase in PGE(2) concentration in the cerebrospinal fluid (CSF), along with the effects of antipyretic drugs on these responses in rats. Zymosan intra-articularly injected at the dose of 0.5 mg did not affect the body core temperature (Tc) compared with saline (control), whereas at doses of 1 and 2 mg, zymosan promoted a flattened increase in Tc and declined thereafter. The dose of 4 mg of zymosan was selected for further experiments because it elicited a marked and long-lasting Tc elevation starting at 3 1/2 h, peaking at 5 1/2 h, and remaining until 10 h. This temperature increase was preceded by a decrease in the tail skin temperature, as well as hyperalgesia and edema in the knee joint. No febrile response was observed in the following days. In addition, zymosan-induced fever was not modified by the sciatic nerve excision. Zymosan increased PGE2 concentration in the CSF but not in the plasma. Oral pretreatment with ibuprofen (5-20 mg/kg), celecoxib (1-10 mg/kg), dipyrone (60-240 mg/kg), and paracetamol (100-200 mg/kg) or subcutaneous injection of dexamethasone (0.25-1.0 mg/kg) dose-dependently reduced or prevented the fever during the zymosan-induced arthritis. Celecoxib (5 mg/kg), paracetamol (150 mg/kg), and dipyrone (120 mg/kg) decreased CSF PGE2 concentration and fever during zymosan-induced arthritis, suggesting the involvement of PGE2 in this response.

Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis

Patients with sepsis have a marked defect in neutrophil migration. Here we identify a key role of Toll-like receptor 2 (TLR2) in the regulation of neutrophil migration and resistance during polymicrobial sepsis. We found that the expression of the chemokine receptor CXCR2 was dramatically down-regulated in circulating neutrophils from WT mice with severe sepsis, which correlates with reduced chemotaxis to CXCL2 in vitro and impaired migration into an infectious focus in vivo. TLR2 deficiency prevented the down-regulation of CXCR2 and failure of neutrophil migration. Moreover, TLR2(-/-) mice exhibited higher bacterial clearance, lower serum inflammatory cytokines, and improved survival rate during severe sepsis compared with WT mice. In vitro, the TLR2 agonist lipoteichoic acid (LTA) down-regulated CXCR2 expression and markedly inhibited the neutrophil chemotaxis and actin polymerization induced by CXCL2. Moreover, neutrophils activated ex vivo by LTA and adoptively transferred into naive WT recipient mice displayed a significantly reduced competence to migrate toward thioglycolate-induced peritonitis. Finally, LTA enhanced the expression of G protein-coupled receptor kinases 2 (GRK2) in neutrophils; increased expression of GRK2 was seen in blood neutrophils from WT mice, but not TLR2(-/-) mice, with severe sepsis. Our findings identify an unexpected detrimental role of TLR2 in polymicrobial sepsis and suggest that inhibition of TLR2 signaling may improve survival from sepsis.

The paraventricular nucleus of the hypothalamus is involved in cardiovascular responses to acute restraint stress in rats

The paraventricular nucleus of the hypothalamus (PVN) has been implicated in several aspects of cardiovascular control. Stimulation of the PVN evokes changes in blood pressure and heart rate. Additionally, this brain area is connected to several limbic structures implicated in behavioral control, as well as to forebrain and brainstem structures involved in cardiovascular control. This evidence indicates that the PVN may modulate cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint is an unavoidable stressor that evokes marked and sustained cardiovascular changes, which are characterized by elevated mean arterial pressure (MAP) and an intense heart rate (HR) increase. We report on the effect of inhibition of PVN synapses on MAP and HR responses evoked by acute restraint in rats. Bilateral microinjection of the nonspecific synaptic blocker cobalt (CoCl2, 1mM/100nl) into the PVN did not change the HR response or the initial peak of the MAP response to restraint stress, but reduced the area under the curve of the MAP response. Moreover, bilateral microinjection of cobalt in areas surrounding the PVN did not change the cardiovascular response to restraint. These results indicate that synapses in the PVN are involved in the neural pathway that controls blood pressure changes evoked by restraint.

The role of neutrophils in severe sepsis

Neutrophils are key effectors of the innate immune response. Reduction of neutrophil migration to infection sites is associated with a poor outcome in sepsis. We have demonstrated a failure of neutrophil migration in lethal sepsis. Together with this failure, we observed more bacteria in both peritoneal exudates and blood, followed by a reduction in survival rate. Furthermore, neutrophils obtained from severe septic patients displayed a marked reduction in chemotactic response compared with neutrophils from healthy subjects. The mechanisms of neutrophil migration failure are not completely understood. However, it is known that they involve systemic Toll-like receptor activation by bacteria and/or their products and result in excessive levels of circulating cytokines/chemokines. These mediators acting together with LPS stimulate expression of iNOS that produces high amounts of NO, which in turn mediates the failure of neutrophil migration. NO reduced expression of CXCR2 on neutrophils and the levels of adhesion molecules on both endothelial cells and neutrophils. These events culminate in decreased endothelium-leukocyte interactions, diminished neutrophil chemotactic response, and neutrophil migration failure. Additionally, the NO effect, at least in part, is mediated by peroxynitrite. In this review, we summarize what is known regarding the mechanisms of neutrophil migration impairment in severe sepsis.

Paraventricular nucleus modulates autonomic and neuroendocrine responses to acute restraint stress in rats

The paraventricular nucleus of the hypothalamus (PVN) has been implicated in several aspects of neuroendocrine and cardiovascular control The PVN contains parvocellular neurons that release the corticotrophin release ha mone (CRH) under stress situations In addition this brain area is connected to several limbic structures implicated in defensive behavioral control as well to forebrain and brainst m structures involved in cardiovascular control Acute restraint is an unavoidable stress situation that evokes corticosterone release as well as marked autonomic changes the latter characterized by elevated mean arterial pressure (MAP) intense heart rate (HR) Increases and decrease in the tail temperature We report the effect of PVN inhibition on MAP and HR responses corticosterone plasma levels and tail temperature response during acute restraint in rats Bilateral microinjection of the nonspecific synaptic blocker CoCl(2) (1 mM/100 nL) into the PVN reduced the pressor response it inhibited the increase in plasma corticosterone concentration as well as the fall in tail temperature associated with acute restraint stress Moreover bilateral microinjection of CoCl(2) into areas surrounding the PVN did not affect the blood pressure hormonal and tail vasoconstriction responses to restraint stress The present results show that a local PVN neurotransmission is involved in the neural pathway that controls autonomic and neuroendocrine responses which are associated with the exposure to acute restraint stress (C) 2010 Elsevier B V All rights reservi.d

Hematopoietic SCT modulates gut inflammation in experimental inflammatory bowel disease

Hematopoietic SCT (HSCT) and high-dose chemotherapy are being explored as therapy for various human refractory immune-mediated conditions, including inflammatory bowel diseases (IBD). Nevertheless, the exact immunological mechanisms by which the BM cells (BMCs) or immunosuppression provide remission from these diseases is not yet clear. In this work, we investigated the role of these therapies in the modulation of gut mucosal inflammation in an experimental model of IBD. Colitis was induced in mice by 2,4,6-trinitrobenzenesulfonic acid and after CY was administered (200 mg/kg) alone (CY group) or followed by BMCs infusion (HSCT group). Animals were followed for 60 days. Both HSCT and CY reduced the histopathological features of colitis significantly. Infused cells were localized in the gut, and a marked decrease of CD4(+) leukocytes in the inflammatory infiltrate on days +7 and +14 and of CD8(+) cells on day +7 was found in both treatments allied to impressive reduction of proinflammatory Th1 and Th17 cytokines. Although chemotherapy alone was the best treatment regarding the induction of immunosuppressive molecules, only HSCT resulted in increased survival rates compared with the control group. Our findings indicate that high-dose CY followed by HSCT is effective in the modulation of mucosal immunity and in accelerating immune reconstitution after BMT, thus providing valuable tools to support the development and understanding of novel therapeutic strategies for IBD. Bone Marrow Transplantation (2010) 45, 1562-1571; doi:10.1038/bmt.2010.6; published online 15 March 2010

The insular cortex modulates cardiovascular responses to acute restraint stress in rats

Acute restraint is an unavoidable stress situation that evokes marked and sustained cardiovascular changes, which are characterized by blood pressure and heart rate increases. In the present study, we tested the hypothesis that insular cortex mediates cardiovascular responses to acute restraint stress in rats. To that purpose, the insular cortex synaptic transmission was inhibited by bilateral microinjection of the nonselective synaptic blocker cobalt chloride (CoCl(2), 1 mM/100 nL). Insular cortex pretreatment with CoCl(2) decreased restraint-evoked pressor and tachycardiac responses, thus indicating an involvement of synapses within the insular cortex on the modulation of cardiovascular responses to restraint stress. The present results indicate that insular cortex synapses exert a facilitatory influence on blood pressure and HR increase evoked by acute restraint stress in rats. Crown Copyright (C) 2010 Published by Elsevier B.V. All rights reserved.

Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model

Aim of the study: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. it comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). Material and methods: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII), and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. Results: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. Conclusions: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Effect of baroreceptor denervation on the autonomic control of arterial pressure in conscious mice

This study evaluated the role of arterial baroreceptors in arterial pressure (AP) and pulse interval (PI) regulation in conscious C57BL mice. Male animals, implanted with catheters in a femoral artery and a jugular vein, were submitted to sino-aortic (SAD), aortic (Ao-X) or carotid sinus denervation (Ca-X), 5 daysprior to the experiments. After basal recording of AP, the lack of reflex bradycardia elicited by administration of phenylephrine was used to confirm the efficacy of SAD, and cardiac autonomic blockade with methylatropine and propranolol was performed. The AP and PI variability were calculated in the time and frequency domains (spectral analysis/fast Fourier transform) with the spectra quantified in low-(LF; 0.25-1Hz) and high-frequency bands (HF; 1-5Hz). Basal AP and AP variability were higher after SAD, Ao-X or Ca-X than in intact mice. Pulse interval was similar among the groups, whereas PI variability was lower after SAD. Atropine elicited a slight tachycardia in control mice but did not change PI after total or partial denervation. The bradycardia caused by propranolol was higher after SAD, Ao-X or Ca-X compared with intact mice. The increase in the variability of AP was accompanied by a marked increase in the LF and HF power of the AP spectra after baroreceptor denervation. The LF and HF power of the PI were reduced by SAD and by Ao-X or Ca-X. Therefore, both sino-aortic and partial baroreceptor denervation in mice elicits hypertension and a remarkable increase in AP variability and cardiac sympathetic tonus. Spectral analysis showed an important contribution of the baroreflex in the power of LF oscillations of the PI spectra. Both sets of baroreceptors seem to be equally important in the autonomic regulation of the cardiovascular system in mice.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamus in vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-alpha-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POW mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 mu g/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POW neurons in the hypothalamus and an increased mRNA expression of these neuropeptides. (C) 2008 Elsevier Inc. All rights reserved.