No description available.

No description available.

No description available.

No / Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex. Cortical spreading depression promotes lesion progression in experimental stroke, and may contribute to the initiation of migraine attacks. The purpose of this study was to investigate the roles of the marked increase of nitric oxide (NO) formation that occurs with CSD. Microdialysis electrodes were implanted in the cortex of anesthetized rats to perform the following operations within the same region: (1) elicitation of CSD by perfusion of high K+ medium; (2) recording of CSD elicitation; (3) application of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME); and (4) recording of dialysate pH changes. The primary effect of L-NAME (0.3 to 3.0 mmol/L in the perfusion medium) was a marked widening of individual CSD wave, resulting essentially from a delayed initiation of the repolarization phase. This change was due to NO synthase inhibition because it was not observed with the inactive isomer D-NAME, and was reversed by L-arginine. This effect did not appear to be linked to the suppression of a sustained, NO-mediated vascular change associated with the superposition of NO synthase inhibition on high levels of extracellular K+. The delayed initiation of repolarization with local NO synthase inhibition may reflect the suppression of NO-mediated negative feedback mechanisms acting on neuronal or glial processes involved in CSD genesis. However, the possible abrogation of a very brief, NO-mediated vascular change associated with the early phase of CSD cannot be ruled out.

Diabetes mellitus is a complex disease characterised by chronic hyperglycaemia due to compromised insulin synthesis and secretion, or decreased tissue sensitivity to insulin, if not all three conditions. Endothelial dysfunction is a common complication in diabetes in which endothelium-dependent vasodilation is impaired. The aim of this study was to examine the involvement of TRPV4 in diabetes endothelial dysfunction. Male Charles River Wistar rats (350-450 g) were injected with 65mg/kg streptozotocin (STZ) intraperitoneally. STZ-injected rats were compared with naïve rats (not injected with STZ) or control rats (injected with 10ml/kg of 20mM citrate buffer, pH 4.0-4.5), if not both. Rats with blood glucose concentrations greater than 16mmol/L were considered to be diabetic. As the results revealed, STZ-diabetic rats showed significant endothelial dysfunction characterised by impaired muscarinic-induced vasodilation, as well as significant impairment in TRPV4-induced vasodilation in aortic rings and mesenteric arteries. Furthermore, STZ-diabetic primary aortic endothelial cells (ECs) showed a significant reduction in TRPV4-induced intracellular calcium ([Ca2+]i) elevation. TRPV4, endothelial nitric oxide synthase (eNOS), and caveolin-1 (CAV-1) were also significantly downregulated in STZ-diabetic primary aortic ECs and were later significantly restored by in vitro insulin treatment. Methylglyoxal (MGO) was significantly elevated in STZ-diabetic rat serum, and nondiabetic aortic rings incubated with MGO (100μM) for 12 hours showed significant endothelial dysfunction. Moreover, nondiabetic primary aortic ECs treated with MGO (100μM) for 5 days showed significant TRPV4 downregulation and significant suppression of 4-α-PDD-induced [Ca2+]i elevation, which was later restored by L-arginine (100μM) co-incubation. Incubating nondiabetic aortic rings with MGO (100μM) for 2 hours induced a spontaneous loss of noradrenaline-induced contractility persistence. Moreover, MGO induced significant [Ca2+]i elevation in Chinese hamster ovary cells expressing rat TRPM8 channels (rTRPM8), which was significantly inhibited by AMTB (1-5μM). Taken together, TRPV4, CAV-1, and eNOS can form a functional complex that is downregulated in STZ-diabetic aortic ECs and restored by insulin treatment. MGO elevation might furthermore contribute to diabetes endothelial dysfunction and TRPV4 downregulation. By contrast, MGO induced the loss of contractility persistence, possibly due to MGO's acting as a TRPM8 agonist.

O óxido nítrico (NO) é um mensageiro químico gerado pela atividade da enzima óxido nítrico sintase (NOS) a qual foi detectada em vários órgãos incluídos os do sistema reprodutor e parece estar envolvido na maturação oocitária. No entanto, há poucos estudos sobre o papel desse sistema em oócitos da espécie bovina. Sabe-se que o NO atua pela via da guanilato ciclase (GC) estimulando a produção do nucleotídeo GMPc, que por sua vez é capaz de influenciar nos níveis de outro nucleotídeo (AMPc) via fosfodiesterases (PDE). O AMPc é um importante elemento da via de sinalização do FSH nos complexos cumulus-oócitos e no controle da maturação oocitária. O objetivo do presente projeto foi investigar a influência do FSH na via do NOS/NO e seus componentes em oócitos bovinos maturados in vitro e o envolvimento das células do cumulus (CC) na via de sinalização. Para tanto, complexos cumulus-oócito (CCO) e oócitos desnudos (OD - maturados sem células do cumulus) foram maturados in vitro por 24h na presença ou ausência de FSH. As amostras foram avaliadas quanto a: 1) taxa da maturação nuclear; 2) níveis de produção de NO; 3) níveis de AMPc e GMPc; 4) abundância relativa de RNAm de NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A e PKA1. O FSH, na concentração de 0,05UI/mL, estimulou positivamente a maturação nuclear em CCO e OD, com 80,6 e 89% de oócitos maturados, respectivamente. Quando comparados diretamente os grupos CCO e OD, o FSH não influenciou as taxas de maturação (71 e 71,3%, p>0,05), nem os níveis de produção de NO (12,8 e 7,4 µM/mL, p>0,05). Os níveis de GMPc em CCO aumentaram após 1 e 3 h de MIV na presença de FSH (266,3 e 187,2 pmol/pool com FSH e 240,5 e 168,5 pmol/pool sem FSH, respectivamente, p<0,05). Após 6 h os níveis de GMPc declinaram de forma mais acentuada no grupo sem FSH (46,3 e 106,9 pmol/pool, com e sem FSH, respectivamente, p<0,05). Os níveis de AMPc em CCO também foram mais elevados na presença de FSH à 1 e 3 h de MIV (7,60 e 7,81 pmol/pool, respectivamente) em comparação com CCO maturados sem FSH (0.30 e 0,76 pmol/pool, respectivamente, p<0,05). Após 6h, os níveis declinaram e foram similares para ambos os grupos (0,43 pmol/pool, p>0,05). Em relação à expressão dos genes selecionados, todos foram detectados nos oócitos (CCO e OD), porém, em células do cumulus, foram detectados apenas PDE5A, ADCY6, ADCY9 e PKA1. Quando observados os resultados do grupo CCO, apenas os genes PKG1, ADCY6 e PDE3A sofreram influência do FSH (p<0,05), apresentando um aumento destes transcritos. No grupo OD, apenas o gene PKG1 sofreu influência do FSH, também apresentando um aumento destes transcritos (p<0,05). Em células do cumulus, os genes ADCY6 e ADCY9 sofreram influência do FSH, sendo que para a ADCY6 provocou um aumentos destes transcritos, e para a ADCY9 provocou uma queda dos mesmos (p<0,05). Em conclusão, o FSH pode exercer influência positiva na maturação nuclear de oócitos bovinos, agindo sobre os níveis de GMPc e AMPc, mas não sobre o NO. O FSH pode influenciar a expressão gênica em oócitos e em células do cumulus de bovinos. / Nitric oxide (NO) is a chemical messenger generated by the nitric oxide synthase (NOS) enzyme, which was detected in several organs including the reproductive system and appears too involved in oocyte maturation. However, there are few studies on the role of this system in bovine oocytes. NO is known to act via guanylate cyclase (GC) stimulating the production of the nucleotide cGMP, which in turn is capable of influencing the levels of another nucleotide, cAMP via phosphodiesterases (PDE). cAMP is an important factor in FSH signaling in cumulus-oocyte complexes (COC) for the control of maturation. The aim of the present work was to investigate the influence of FSH on the NOS/NO pathway and its components in bovine oocytes matured in vitro and the involvement of cumulus cells (CC) in the signaling pathway. COC and denuded oocytes (DO - matured without cumulus cells) were matured in vitro for 24 h with or without FSH. Samples were assessed for: 1) maturation rate; 2) levels of NO production; 3) levels of cGMP and cAMP; 4) relative abundance for mRNA of NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A and PKA1. FSH positively stimulated oocyte maturation at 0.05UI/mL concentration for both COC and OD (80.6 and 89% maturation rates, respectively). When COC and OD were compared directly, FSH did not affect maturation rates (71 and 71.3%, p>0.05) nor NO production levels (12,8 and 7,4 µM/mL), p>0.05). cGMP levels increased after 1 and 3 h in vitro maturation (IVM) with FSH (266.3 and 187.2 pmol/pool with FSH and 240.5 and 168.5 pmol/pool without FSH, respectively, p<0.05). After 6 h IVM, cGMP levels in COC declined more in the group cultured with FSH (46.3 and 106.9 pmol/pool, with and without FSH, respectively, p<0.05). cAMP levels in COC were also increased in the presence of FSH at 1 and 3 h IVM (7.60 and 7.81 pmol/pool, respectively) in comparison to COC cultured without the hormone (0.30 and 0.76 pmol/pool, respectively, p<0.05). After 6 h, the levels declined and were similar for both groups (0.43 and 0.02 pmol/pool, p>0.05). Regarding mRNA expression for the selected genes, all of them were detected in oocytes, but only four of them were detected in cumulus cells: PDE5A, ADCY6, ADCY9 and PKA1. For COC only PKG1, ADCY6 and PDE3A were influenced by FSH (p<0.05), with an increase in transcript relative abundance, For DO, only PKG1 was influenced by FSH and also showed an increase in these transcripts (p<0.05). In cumulus cells, ADCY6 and ADCY9 were affected by FSH, with an increase for ADCY6 and a decrease in ADCY9 transcripts (p<0.05). In conclusion, FSH may positively influence nuclear maturation, acting on cGMP and cAMP levels, but not on NO. FSH may also influence gene expression in bovine oocytes and cumulus cells.

Obesity is associated with chronic inflammation and increased risk of developing cardiovascular disease (CVD). Obesity is also associated with nitric oxide (NO)-mediated vascular endothelial dysfunction (VED), an independent predictor of increased CVD risk in humans. Pro-inflammatory cytokines secreted by the adipose tissue, such as TNF-Α, may contribute to VED through promotion of insulin resistance or directly through a reduction in endothelial NO synthase (eNOS) expression and/or phosphorylation. Sodium salicylate (Na-Sal) is a non-acetylated aspirin that inhibits the pro-inflammatory transcription factor nuclear factor-ΚB (NF-ΚB) and activates the cellular metabolism regulator AMP-activated protein kinase (AMPK). AMPK is a known activator of eNOS. We tested the hypothesis that Na-Sal increases eNOS expression/phosphorylation in TNFΑ-stimulated endothelial cells through an AMPK-dependent mechanism. Human aortic endothelial cells (HAECs) incubated in vitro with TNF-Α (10 ng/ml, 2 hrs) demonstrated decreased (vs. control) expression (via Western blotting) of eNOSser1177 phosphorylation (n=8; PThr172 phosphorylation (n=8, Pser1177 phosphorylation (vs. control, n=7; P=0.14) and AMPKThr172 phosphorylation (vs. control, n=9; P=0.42). The AMPK activator AICAR prevented eNOSser1177 phosphorylation down-regulation by TNF-Α in a manner similar to Na-Sal (n=2, P=0.839). Co-treatment with the AMPK inhibitor compound C (10 μM, 30 min) abolished the ability of Na-Sal to prevent down-regulation of eNOSser1177 phosphorylation by TNF-Α (vs. control, n=3; Pser1177 in endothelial cells in part through AMPK.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells identified in mice as Gr-1+CD11b+ cells with the ability to inhibit T cell function. MDSC are emerging as important regulators of T cell mediated immune responses. Current paradigm suggests that despite heterogeneity, all Gr-1+CD11b+ cells are suppressive when exposed to inflammatory stimuli. In vitro evaluation shows MDSC from multiple tissue sites have suppressive activity, and in vivo inhibition of MDSCenhances T cell function. However, the relative capacity of MDSC present at localized inflammatory sites or in peripheral tissues to suppress T cell responses in vivo has not been directly evaluated. Using a tissue specific acute inflammatory prostatitis model, we demonstrate that MDSC inhibition of CD8+ T-cell proliferation is restricted to the inflammatory site.Further, MDSC from inflammatory sites possess immediate capacity to inhibit T-cell function, whereas those isolated from peripheral tissues (spleens and liver) were not suppressive without activation of iNOS by exposure to IFN-_.Using two mouse models of prostate cancer, we extend these findings to thetumor micro-environment. During a chronic inflammatory response induced by tumorgrowth, we show Gr-1+CD11b+ cells from the tumor site possess immediate capacity toregulate effector T cell function whereas those from the spleen do not. In both tumormodels and in our prostatitis model, long term culture of activated T cells with splenicGr-1+CD11b+ cells converted precursor cells into functional MDSC during standard in vitro suppression assays. These data highlight the importance of MDSC in the prostate, and demonstrate the function of MDSC during a localized inflammatory response isrestricted to the site of an ongoing immune responseGrowing evidence suggests that prostatitis associated with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is mediated in part by the loss of T cell and B cell tolerance to prostate antigens. Clinical data demonstrates the presence of T cell proliferative responses to prostate auto-antigens in CP/CPPS patients. However, the mechanisms leading to this loss of tolerance are not clearly understood, largely because of a lack of available animal models. We report the development of a new mouse model for the study of chronic prostate inflammation (CPI), the Prostate Ovalbumin Expressing Transgenic-3 (POET-3) model. Adoptive transfer of antigen specific OT-I T cells induces CPI characterized by infiltration of exogenous (OT-I) and endogenous T cells into the prostate persisting as long as 45 days after transfer. In vitro and in vivo data demonstrate inflammation induced loss of T cell tolerance to prostate auto-antigens. Auto-antibody responses to prostate antigens were detected in POET-3 mice after induction of CPI. These data have important therapeutic implications for treatment of CPI.

Abstract not available

Duchenne Muscular Dystrophy (DMD) is a fatal condition occurring in approximately 1 in 3500 male births and is due to the lack of a protein called dystrophin. Initially DMD was considered a skeletal myopathy, but the pathology and consequences of cardiomyopathy are being increasingly recognised. Fibrosis, resulting from continual cycles of degeneration of the muscle tissues followed by inadequate regeneration of the muscles, is progressive in both cardiac and skeletal dystrophic muscle. In the heart fibrosis interferes with contractility and rhythm whereas it affects contractile function and causes contractures in skeletal muscles. This study utilised the mdx mouse which exhibits a pathological loss of muscle fibres and fibrosis characteristic of DMD, to examine a range of mechanisms that can influence muscle function and fibrosis. Ageing and workload both appear to contribute to the development of dystrophic features in cardiac and skeletal muscle of the mdx mouse. Therefore the effect of eccentric exercise on cardiac and skeletal muscle was examined in older mdx mice. Mice ran in 30 minute sessions for five months, 5 days per week. Downhill treadmill running did not exacerbate the contractile function or fibrosis of the mdx heart or the EDL, SOL or diaphragm muscles suggesting that cytokines influence function and fibrosis to a greater extent than workload alone. The role of the cytokine TGF-beta was examined by treating mdx mice with the TGF-beta antagonist pirfenidone at 0.4, 0.8 or 1.2 per cent in drinking water for six months. Pirfenidone improved cardiac contractility (P<0.01) and coronary flow (P<0.05), to levels comparable to control mice, despite no reduction in cardiac fibrosis. Pirfenidone did not reduce fibrosis or improve function in skeletal muscle. A deficiency of neuronal nitric oxide synthase (nNOS) in DMD and mdx mice causes a lowered production of nitric oxide indicating that the substrate of nNOS, l-arginine, may be beneficial to cardiac and skeletal muscle function in mdx mice. Oral l-arginine (5 mg/g bw) improved cardiac contractility, coronary flow and reduced cardiac fibrosis (P<0.05) without improving skeletal muscle function or fibrosis. In contrast, 10 mg/g bw l-arginine improved cardiac function and coronary flow (P<0.01), despite also elevating cardiac collagen. This increment in collagen was prevented by co-administration of prednisone. The experiments described in this dissertation reveal for the first time that pharmacological treatments in mdx mice can improve cardiac structure and function. Further elucidation of the optimum time and doses of such treatments may result in future pharmacological treatments to improve cardiac function and fibrosis in DMD.