Sympathetic hyperactivity, increased tyrosine hydroxylase and exaggerated corpus cavernosum relaxations associated with oxidative stress plays a major role in the penis dysfunction in townes sickle cell mouse

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOSickle cell disease patients display priapism that may progress to erectile dysfunction. However, little is known about the pathophysiological alterations of corpus cavernosum in sickle cell disease. Objective Thus, this study aimed to evaluate the functional and molecular alterations of sympathetic machinery and nitric oxide-cyclic guanosine monophosphate signaling pathway in Townes transgenic sickle cell disease mice. Methods Concentration-response curves to contractile (phenylephrine) and relaxant agents (acetylcholine and sodium nitroprusside) were obtained in corpus cavernosum strips from sickle and C57BL/6 (control) mice. Neurogenic contractions and nitrergic relaxations were obtained using electrical-field stimulation. Measurements of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), phosphodiesterase-5 (PDE5) and a alpha(1A)-, alpha(1B)-and alpha(1D)-adrenoceptor mRNA expressions and reactive-oxygen species were performed. Tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expressions in cavernosal tissues were also measured. Results The neurogenic contractions were higher in the sickle cell disease group, in association with elevated tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expression, as well as increased tyrosine hydroxylase mRNA expression. Likewise, phenylephrine-induced contractions were greater in the sickle mice, whereas a alpha(1A)-, alpha(1B)-and alpha(1D)-adrenoceptor mRNA expression remained unchanged. Cavernosal relaxations to acetylcholine, sodium nitroprusside and EFS were higher in sickle mice, accompanied by decreased eNOS and nNOS, along with lower PDE5 mRNA expression. An increase of about 40% in reactive-oxygen species generation in corpus cavernosum from sickle mice was also detected. Conclusion Our study shows that decreased nitric oxide bioavailability in erectile tissue due to increased oxidative stress leads to both sympathetic hyperactivity and dysregulation of nitric oxide signaling in corpus cavernosum from Townes sickle mice.Sickle cell disease patients display priapism that may progress to erectile dysfunction. However, little is known about the pathophysiological alterations of corpus cavernosum in sickle cell disease. Objective Thus, this study aimed to evaluate the functional and molecular alterations of sympathetic machinery and nitric oxide-cyclic guanosine monophosphate signaling pathway in Townes transgenic sickle cell disease mice. Methods Concentration-response curves to contractile (phenylephrine) and relaxant agents (acetylcholine and sodium nitroprusside) were obtained in corpus cavernosum strips from sickle and C57BL/6 (control) mice. Neurogenic contractions and nitrergic relaxations were obtained using electrical-field stimulation. Measurements of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), phosphodiesterase-5 (PDE5) and a alpha(1A)-, alpha(1B)-and alpha(1D)-adrenoceptor mRNA expressions and reactive-oxygen species were performed. Tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expressions in cavernosal tissues were also measured. Results The neurogenic contractions were higher in the sickle cell disease group, in association with elevated tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expression, as well as increased tyrosine hydroxylase mRNA expression. Likewise, phenylephrine-induced contractions were greater in the sickle mice, whereas a alpha(1A)-, alpha(1B)-and alpha(1D)-adrenoceptor mRNA expression remained unchanged. Cavernosal relaxations to acetylcholine, sodium nitroprusside and EFS were higher in sickle mice, accompanied by decreased eNOS and nNOS, along with lower PDE5 mRNA expression. An increase of about 40% in reactive-oxygen species generation in corpus cavernosum from sickle mice was also detected. Conclusion Our study shows that decreased nitric oxide bioavailability in erectile tissue due to increased oxidative stress leads to both sympathetic hyperactivity and dysregulation of nitric oxide signaling in corpus cavernosum from Townes sickle mice.1112FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2013/19781-2; 2014/00984-

Pain assessment in surgical patients with impaired cognition

Pain is considered the 5th vital sign and its measurement/assessment and records are required and must be systematic. Ineffective pain management involves complications in clinical status of patients, longer hospitalization times and higher costs with health. In the surgical patient with impaired cognition, hetero measurements should be made, based on behavioural and physiological indicators. We used to determine the efficacy and efficiency of the Observer Scale, the Abbey Pain Scale and Pain Assessment in Advanced Dementia (PAINAD). Our study is an applied, non-experimental, quantitative, descriptive and analytical research. The data collection instrument consisted of patients’ sociodemographic and clinical data, the Observer Scale, the Abbey Pain Scale (Rodrigues, 2013) and PAINAD (Batalha et al., 2012). We assessed pain at an early phase and 45 minutes after an intervention for its relief. The sample is non-probabilistic for convenience, consisting of 76 surgical patients with impaired cognition, admitted to the surgery services of a central hospital, aged between 38 and 96 years. There was a positive correlation between the results of the three scales, most evident in the initial evaluation. Pain intensity in the same patient is higher when assessed with PAINAD (OM = 2.16) and lower when assessed with the Observer Scale (OM = 1.78). The most effective and efficient scale is PAINAD. Due to the small sample size, we suggest confirmatory studies so that the results can be generalized

Association Of Nitric Oxide Synthase And Matrix Metalloprotease Single Nucleotide Polymorphisms With Preeclampsia And Its Complications.

Preeclampsia is one of the leading causes of maternal and neonatal morbidity and mortality in the world, but its appearance is still unpredictable and its pathophysiology has not been entirely elucidated. Genetic studies have associated single nucleotide polymorphisms in genes encoding nitric oxide synthase and matrix metalloproteases with preeclampsia, but the results are largely inconclusive across different populations. To investigate the association of single nucleotide polymorphisms (SNPs) in NOS3 (G894T, T-786C, and a variable number of tandem repetitions VNTR in intron 4), MMP2 (C-1306T), and MMP9 (C-1562T) genes with preeclampsia in patients from Southeastern Brazil. This prospective case-control study enrolled 77 women with preeclampsia and 266 control pregnant women. Clinical data were collected to assess risk factors and the presence of severe complications, such as eclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome. We found a significant association between the single nucleotide polymorphism NOS3 T-786C and preeclampsia, independently from age, height, weight, or the other SNPs studied, and no association was found with the other polymorphisms. Age and history of preeclampsia were also identified as risk factors. The presence of at least one polymorphic allele for NOS3 T-786C was also associated with the occurrence of eclampsia or HELLP syndrome among preeclamptic women. Our data support that the NOS3 T-786C SNP is associated with preeclampsia and the severity of its complications.10e013669

Urinary bladder dysfunction in transgenic sickle cell disease mice

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOBackground Urological complications associated with sickle cell disease (SCD), include nocturia, enuresis, urinary infections and urinary incontinence. However, scientific evidence to ascertain the underlying cause of the lower urinary tract symptoms in SCD is lacking. Objective Thus, the aim of this study was to evaluate urinary function, in vivo and ex vivo, in the Berkeley SCD murine model (SS). Methods Urine output was measured in metabolic cage for both wild type and SS mice (25-30 g). Bladder strips and urethra rings were dissected free and mounted in organ baths. In isolated detrusor smooth muscle (DSM), relaxant response to mirabegron and isoproterenol (1 nM-10 mu M) and contractile response to (carbachol (CCh; 1 nM-100 mu M), KCl (1 mM-300mM), CaCl2 (1 mu M-100mM), alpha,beta-methylene ATP (1, 3 and 10 mu M) and electrical field stimulation (EFS; 1-32 Hz) were measured. Phenylephrine (Phe; 10nM-100 mu M) was used to evaluate the contraction mechanism in the urethra rings. Cystometry and histomorphometry were also performed in the urinary bladder. Results SS mice present a reduced urine output and incapacity to produce typical bladder contractions and bladder emptying (ex vivo), compared to control animals. In DSM, relaxation in response to a selective beta 3-adrenergic agonist (mirabegron) and to a non-selective beta-adrenergic (isoproterenol) agonist were lower in SS mice. Additionally, carbachol, alpha,beta-methylene ATP, KCl, extracellular Ca2+ and electrical-field stimulation promoted smaller bladder contractions in SS group. Urethra contraction induced by phenylephrine was markedly reduced in SS mice. Histological analyses of SS mice bladder revealed severe structural abnormalities, such as reductions in detrusor thickness and bladder volume, and cell infiltration. Conclusions Taken together, our data demonstrate, for the first time, that SS mice display features of urinary bladder dysfunction, leading to impairment in urinary continence, which may have an important role in the pathogenesis of the enuresis and infections observed the SCD patients.Urological complications associated with sickle cell disease (SCD), include nocturia, enuresis, urinary infections and urinary incontinence. However, scientific evidence to ascertain the underlying cause of the lower urinary tract symptoms in SCD is lacking. Objective Thus, the aim of this study was to evaluate urinary function, in vivo and ex vivo, in the Berkeley SCD murine model (SS). Methods Urine output was measured in metabolic cage for both wild type and SS mice (25-30 g). Bladder strips and urethra rings were dissected free and mounted in organ baths. In isolated detrusor smooth muscle (DSM), relaxant response to mirabegron and isoproterenol (1 nM-10 mu M) and contractile response to (carbachol (CCh; 1 nM-100 mu M), KCl (1 mM-300mM), CaCl2 (1 mu M-100mM), alpha,beta-methylene ATP (1, 3 and 10 mu M) and electrical field stimulation (EFS; 1-32 Hz) were measured. Phenylephrine (Phe; 10nM-100 mu M) was used to evaluate the contraction mechanism in the urethra rings. Cystometry and histomorphometry were also performed in the urinary bladder. Results SS mice present a reduced urine output and incapacity to produce typical bladder contractions and bladder emptying (ex vivo), compared to control animals. In DSM, relaxation in response to a selective beta 3-adrenergic agonist (mirabegron) and to a non-selective beta-adrenergic (isoproterenol) agonist were lower in SS mice. Additionally, carbachol, alpha,beta-methylene ATP, KCl, extracellular Ca2+ and electrical-field stimulation promoted smaller bladder contractions in SS group. Urethra contraction induced by phenylephrine was markedly reduced in SS mice. Histological analyses of SS mice bladder revealed severe structural abnormalities, such as reductions in detrusor thickness and bladder volume, and cell infiltration. Conclusions Taken together, our data demonstrate, for the first time, that SS mice display features of urinary bladder dysfunction, leading to impairment in urinary continence, which may have an important role in the pathogenesis of the enuresis and infections observed the SCD patients108115FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP [2008/57441-0]CNPq [481761/2008-0]sem informaçãosem informaçã

Papel das fibras aferentes primarias sensiveis a capsaicina no recrutamento de neutrofilos para as vias aereas e na neutropoiese

Orientador: Edson AntunesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: O tratamento neonatal com capsaicina em ratos acarreta aumento da inflamação alérgica pulmonar, sugerindo um papel protetor para as fibras C nas vias aéreas (MEDEIROS et ai, 2001). O objetivo deste trabalho foi investigar os mecanismos responsáveis pelo aumento do influxo neutrofílico pulmonar em ratos desprovidos de neuropeptídeos pelo tratamento neonatal com capsalclna, sensibilizados e desafiados com ovalbumina (OVA) na fase adulta. Os animais receberam capsaicina (30 mg/Kg, s.c.) no segundo dia de vida, sendo os protocolos experimentais realizados nos animais na fase adulta dos mesmos (60- 70 dias de vida). A sensibilização com OVA foi realizada pela injeção subcutânea de 200 ~g de OVA adsorvida em 8 mg de AI (OHh, O desafio antigênico com OVA (1 mg) foi realizado 14 dias após a sensibilização. A contagem de células no LBA, sangue periférico e medula óssea foi realizada 6h após o desafio com OVA. Nos animais desafiados com OVA observamos um aumento no número de neutrófilos no LBA, sendo este aumento significativamente maior em animais pré-tratados com capsaicina. Em ambos os grupos, controle e capsaicina, observamos redução do número de neutrófilos no sangue periférico de ratos desafiados com OVA, sendo esta reduçao maior no grupo capsaicina. Na medula óssea, observamos aumento significativo das formas imaturas e maduras de neutrófilos em ratos desafiados com OVA, não foi observado diferenças estatísticas entre o grupo controle e capsaicina. Empregando o modelo de adesão in vitra, nossos resultados mostraram que os neutrófilos sanguíneos obtidos de ratos tratados com capsaicina têm a mesma capacidade de aderir em resposta ao fMLP (0.1 ~M) ou PMA (1 ~M) em placas recobertas com soro ou fibronectina. Além disso, neutrófilos obtidos de animais controle ou pré-tratados com capsaicina são XXV1ll capazes de migrar in vitro em resposta ao fMLP (10 flM), PAF (0.1 j.1M) e a substância P (50 j.1M) de maneira semelhante. Observamos um aumento significativo nos níveis de TNF-a, IL-5, IL-10 e LTB4 no LBA dos ratos desafiados com OVA e pré-tratados com capsaicina. Nos animais tratados com capsaicina observamos um aumento significativo na expressão do RNAm para CINC-3 e redução do RNAm para CINC-2 no pulmão após o desafio com OVA, quando comparado com o seu respectivo controle. Macrófagos alveolares obtidos de animais tratados com capsaicina foram capazes de gerar a mesma quantidade de ânion superóxido, nitrito e TNF-a (basal e estimulada) quando comparado com o grupo controle. O tratamento crônico dos ratos com o composto 48/80 preveniu o aumento do influxo de neutrófilos no grupo de animais tratados com capsaicina (desafiados com OVA) e restaurou os níveis de TNF-a aos valores encontrados no grupo controle. No conjunto, estes resultados sugerem que o tratamento neonatal com capsaicina resulta em "up-regulação" dos mastócitos, que, uma vez ativados pela exposição ao antígeno, passam a liberar grandes quantidades de mediadores inflamatórios, principalmente do TNF-a e a CINC-3, amplificando desta forma aumento do influxo de neutrófilos nas vias aéreas destes animais. Paralelamente, observamos que ratos pré-tratados com capsaicina apresentam aumento no número basal de neutrófilos imaturos e maduros na medula óssea, quando comparado aos animais controle. Este aumento está associado à elevação da expressão da PPT-I e do receptor NK1 na medula óssea, sugerindo que estas proteínas estão "upreguladas" em animais desprovidos de neuropeptídeos pelo tratamento neonatal com capsaicinaAbstract: This study was undertaken to clarify the mechanisms by which C-fiber degeneration at neonatal stages exacerbates the inflammatory responses of rat airways. Rats were treated with capsaicin at neonatal stag es and immunized with ovalbumin (OVA) at adult ages. Challenge of capsaicin pretreated rats with OVA promoted a higher influx of neutrophils in bronchoalveolar lavage (BAL) fluid compared with the vehicle group. No significant differences were found for the other cell types. The increased adhesion of fMLP (0.1 ~M) and PMA (1 ~M)-treated neutrophils to fibronectin-coated wells did not differ among vehicle- and capsaicinpretreated rats. Additionally, fMLP (1O ~M), PAF-(0.1 ~M) and substance P (50~M) induced a significant neutrophil chemotaxis, but no differences were found among vehicle and capsaicin groups. Increased levels of TNF-a, IL-5, IL-10 and LTB4 in BAL fluid as well as higher expression of CINC-3 in lung homogenates were detected in the capsaicin group compared with vehicle group. In the capsaicin group, chronic treatment with compound 48/80 restored the TNF-a levels to control values and prevented the neutrophil influx in BAL fluid. The enhanced production of TNF-a, superoxide anion and nitrite by isolated alveolar macrophages in response to LPS (3 ~g/ml), PMA (10 nM) and/or zymosan (100 particles/cell) did not differ between vehicle- and capsaicin-pretreated rats. In conclusion, chronic neuropeptide depletion promoted by neonatal capsaicin treatment upregulates airways mast cells, which upon activation by antigen at adult ages, release large amounts of cytokines such as TNF-a and CINC-3 that accounts for the massive airways neutrophil infiltration. Parallel to this, we observed that capsaicinXXX pretreated rats present higher numbers of both immature and mature neutrophils in bone marrow, compared with control rats. This was accompanied by higher expression of mRNA for PPT-I and tackynin-NK1 receptor in bone marrow, suggesting that these proteins are up-regulated in rats chronically depleted of neuropeptides by neonatal capsaicin treatmentDoutoradoFarmacologiaDoutor em Farmacologi

Participação do oxido nitrico na inflamação aguda induzida pela enterotoxina estafilococica do tipo B em camundongos

Orientador: Edson AntunesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Neste trabalho investigamos a participação do NO no edema de pata, permeabilidade vascular e influxo de neutrófilos para cavidade peritoneal induzidos pela enterotoxina estafilocócica tipo B (SEB) em camundongos. Para tanto, utilizamos inibidores não-seletivos (L- NAME) e seletivos (aminoguanidina e 7 -nitroindazol) da NO-sintase, assun como a dexametasona (inibidor da indução da iNOS). Nossos resultados mostraram que o L-NAME (7-28 mg!kg, i.v.) e aminoguanidina (25-75 mg!kg, i.v.) reduziram significativamente o edema de pata induzido pela SEB (25 ~g/pata). A administração subplantar de tais inibidores também causou significante redução do edema de pata. Por outro lado, apenas as maiores doses destes inibidores (administrados pela via endovenosa) foram capazes de inibir de maneira significativa, o extrayasamento de proteínas induzido pela toxina. O D- NAME (enântiomero inativo), administrado local ou sistemicamente, não alterou a resposta edematogênica induzida pela SEB. A inibição do edema de pata causada pelo L-NAME e aminoguanidina foi revertida pelo iloprost (análogo da prostaciclina), sugerindo que o efeito inibitório destes inibidores deve-se, provavelmente, à redução de fluxo sangiiíneo local como conseqüência da inibição do NO. Aadministração sistêmica do L-NAME (14-56 mg!kg), aminoguanidina (25-75 mg!kg) ou dexametasona (0,25-1,0 mg!kg) atenuou o influxo de neutrófilos induzido pela SEB em cavidades peritoneais com número de macrófagos normais. Em cavidades com o número de macrófagos aumentado, os inibidores foram menos eficazes em reduzir o influxo dessas células. O D-NAME não afetou o influxo de neutrófilos induzido pela SEB nas duas cavidades estudadas. Os resultados da medida da atividade da NOS mostraram que a nNOS do cérebro foi significativamente inibida pelo L-NAME 4 (28 mg/kg) e 12 h (14-56 mg/kg) após administração endovenosa deste inibidor. A aminoguanidina (75 mg/kg, i.v.) reduziu a atividade da nNOS em 4 h, mas não em 12 h após a administração sistêmica. O 7-nitroindazol (5-15 mg/kg) não modificou a nNOS em quaisquer das doses utilizadas. A atividade da iNOS foi inibida significativamente pelo L-NAME (14-56 mg/kg, i.v., 12 h) e aminoguanidina (25-75 mg/kg, i.v., 12 h). Concluímos que o NO é um mediador importante na resposta edematogênica e no influxo de neutrófilos induzidos pela SEB. A síntese do NO, no sítio inflamatório, parece envolver a cNOS na fase inicial (4 h) e a iNOS na fase de mais tardia (12 h)Abstract: In this study we attempted to investigate the role of nitric oxide (NO) on the inflammatory responses (hindpaw oedema, vascular permeability and neutrophil migration) induced by the staphylococcal enterotoxin type B (SEB) in mouse. To achieve this, both non-selective (L-NAME) and selective (aminoguanidine and 7-nitroindazole) NO-synthase (NOS) inhibitors as well as dexamethasone (iNOS induction inhibitor) were used. Our results showed that L-NAME (7-28 mg/kg, i.v.) and aminoguanidine (25-75 mg/kg, i.v.) significantly reduced SEB-induced paw oedema. Similar results were observed when these inhibitors were administered intraplantarly. The inactive enantiomer D-NAME had no effect on the SEB-induced oedema formation. The inhibition of paw oedema by both L- NAME and aminoguanidine were significantly reversed by the local . administration of the prostacyclin analogue iloprost. This suggest that these inhibitors probably acts by reducting regional blood flow. The compound 7¬nitroindazole, a substance which preferentially inhibit nNOS, had no effect on the exsudation and paw oedema induced by SEB. L-NAME and aminoguanidine as well as dexamethasone significant reduced neutrophil accumulation induced. by SEB into mouse peritonenl cavity. In thioglycollate-pretreated cavities, a condition where the number of macrophages is increased, L-NAME and aminoguanidine caused a smaller inhibition of SEB-induced neutrophil migration, as compared to naive cavities. D-NAME had no effect on the neutrophil migration induced by SEB, either in non-stimulated or thioglycollate-treated cavities. In addition, bNOS activity was reduced by L-NAME at 4 h (28 mg/kg) and 12 h (14-56 mg/kg) after endovenous administration, while aminoguanidine (75 mg/kg, i. v.) was able to reduce the nNOs activity only when this inhibitor was injected 4 h before. The nNOS activity was unchanged by 7 -nitroindazole. The activity of iNOS was significant1y inhibited by L-NAME (14-56 mg/kg, i.v., 12 h) and aminoguanidine (25-75 mg/kg, i.v., 12 h).In conclusion, our results indicate that NO modulate both oedema and neutrophil migration induced by SEB. We suggest that NO involved in inflammatory response was produced by cNOS in the early phase (4 h) and iNOS during the sustained phase (12 h)MestradoFarmacologiaMestre em Ciências Médica

Newer Aspects Of The Pathophysiology Of Sickle Cell Disease Vaso-occlusion.

Sickle cell disease is an inherited disorder of hemoglobin (Hb) synthesis, caused by a single nucleotide substitution (GTG>GAG) at the sixth codon of the beta-globin gene, leading to the production of a defective form of Hb, Hb S. When deoxygenated, Hb S polymerizes, damaging the sickle erythrocyte and it is this polymerization that is the primary indispensable event in the molecular pathogenesis of sickle cell disease. Hb S polymerization results in a series of cellular alterations in red cell morphology and function that shorten the red cell life span and leads to vascular occlusion. Sickle cell disease vaso-occlusion is now known to constitute a complex multifactorial process characterized by recurrent vaso-occlusion, ischemia-reperfusion injury, and oxidative stress with consequent vascular endothelial cell activation that induces a chronic inflammatory state in sickle cell disease individual and is propagated by elevated levels of circulating inflammatory cytokines. Activation of the endothelium results in the induction of endothelial adhesion molecule expression that mediates red and white cell adhesion to the vessel wall and the formation of heterocellular aggregates, followed by secondary red cell trapping, all of which contribute to reduced blood flow and eventually obstruction of the micro-circulation. Reduced nitric oxide bioavailability, caused principally by its consumption by cell-free Hb, liberated during intravascular hemolysis, contributes to this process by facilitating vasoconstriction and adhesion molecule activity.331-1