RESUMO
The Transient Receptor Potential Ankyrin 1 (TRPA1) cation channel expressed on capsaicin-sensitive afferents, immune and endothelial cells is activated by inflammatory mediators and exogenous irritants, e.g., endotoxins, nicotine, crotonaldehyde and acrolein. We investigated its involvement in acute and chronic pulmonary inflammation using Trpa1 gene-deleted (Trpa1-/-) mice. Acute pneumonitis was evoked by intranasal Escherichia coli endotoxin (lipopolysaccharide: LPS) administration, chronic bronchitis by daily cigarette smoke exposure (CSE) for 4 months. Frequency, peak inspiratory/expiratory flows, minute ventilation determined by unrestrained whole-body plethysmography were significantly greater, while tidal volume, inspiratory/expiratory/relaxation times were smaller in Trpa1-/- mice. LPS-induced bronchial hyperreactivity, myeloperoxidase activity, frequency-decrease were significantly greater in Trpa1-/- mice. CSE significantly decreased tidal volume, minute ventilation, peak inspiratory/expiratory flows in wildtypes, but not in Trpa1-/- mice. CSE remarkably increased the mean linear intercept (histopathology), as an emphysema indicator after 2 months in wildtypes, but only after 4 months in Trpa1-/- mice. Semiquantitative histopathological scores were not different between strains in either models. TRPA1 has a complex role in basal airway function regulation and inflammatory mechanisms. It protects against LPS-induced acute pneumonitis and hyperresponsiveness, but is required for CSE-evoked emphysema and respiratory deterioration. Further research is needed to determine TRPA1 as a potential pharmacological target in the lung.
Assuntos
Bronquite Crônica/fisiopatologia , Fumar Cigarros/efeitos adversos , Pneumonia/fisiopatologia , Canal de Cátion TRPA1/metabolismo , Animais , Bronquite Crônica/induzido quimicamente , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Feminino , Lipopolissacarídeos/toxicidade , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Peroxidase/metabolismo , Pletismografia Total , Pneumonia/induzido quimicamente , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Testes de Função Respiratória , Canal de Cátion TRPA1/genéticaRESUMO
Obesity presents a growing public health problem. Therefore the analysis of body composition is important in clinical practice as well as in animal research models of obesity; hence precise methods for the assessment of body fat would be essential. We aimed to evaluate in vivo abdominal microcomputed tomography scan restricted to the L1-L3 region [micro-CT(L1-L3)], a skinfold thickness-based method (STM), and postmortem body composition analysis (PMA) with regard to whole body micro-CT scan in rats. Male Wistar rats of different age groups (from 3 to 24 mo) and nutritional states (normally fed, high-fat diet-induced obese, and calorie-restricted) were used. The fat percentage was determined with micro-CT(L1-L3) and whole body scan in anesthetized rats. Their skinfold thickness was measured in five locations with a Lange caliper. Wet weights of epididymal and retroperitoneal fat pads were determined via PMA. With regard to fat mass, the strongest correlation was observed between abdominal and whole body micro-CT. The other methods showed weaker associations with whole body micro-CT and with each other. Micro-CT(L1-L3) and PMA showed similar age-associated increase in fat mass between 3 and 18 mo. Micro-CT(L1-L3), STM, and PMA were efficient to detect differences in fat mass values in groups of different nutritional states. Micro-CT(L1-L3) appears to be a useful method for body fat assessment in rats with reduced scanning time. In rats, STM may also be a useful, low priced, noninvasive, and simple in vivo technique to assess obesity. NEW & NOTEWORTHY Body fat of rats assessed by in vivo abdominal microcomputed tomography of the L1-L3 region strongly correlates with values determined by whole body scan. Therefore, it is a useful method for fat assessment with reduced scanning time. Skinfold thickness measurement is an in vivo technique to assess progression of obesity in rats.
Assuntos
Gordura Intra-Abdominal/diagnóstico por imagem , Estado Nutricional , Dobras Cutâneas , Fatores Etários , Animais , Composição Corporal , Masculino , Ratos Wistar , Microtomografia por Raio-XRESUMO
Cigarette smoke-triggered inflammatory cascades and consequent tissue damage are the main causes of chronic obstructive pulmonary disease (COPD). There is no effective therapy and the key mediators of COPD are not identified due to the lack of translational animal models with complex characterization. This integrative chronic study investigated cardiopulmonary pathophysiological alterations and mechanisms with functional, morphological and biochemical techniques in a 6-month-long cigarette smoke exposure mouse model. Some respiratory alterations characteristic of emphysema (decreased airway resistance: Rl; end-expiratory work and pause: EEW, EEP; expiration time: Te; increased tidal mid-expiratory flow: EF50) were detected in anaesthetized C57BL/6 mice, unrestrained plethysmography did not show changes. Typical histopathological signs were peribronchial/perivascular (PB/PV) edema at month 1, neutrophil/macrophage infiltration at month 2, interstitial leukocyte accumulation at months 3-4, and emphysema/atelectasis at months 5-6 quantified by mean linear intercept measurement. Emphysema was proven by micro-CT quantification. Leukocyte number in the bronchoalveolar lavage at month 2 and lung matrix metalloproteinases-2 and 9 (MMP-2/MMP-9) activities in months 5-6 significantly increased. Smoking triggered complex cytokine profile change in the lung with one characteristic inflammatory peak of C5a, interleukin-1α and its receptor antagonist (IL-1α, IL-1ra), monokine induced by gamma interferon (MIG), macrophage colony-stimulating factor (M-CSF), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) at months 2-3, and another peak of interferon-γ (IFN-γ), IL-4, 7, 13, 17, 27 related to tissue destruction. Transient systolic and diastolic ventricular dysfunction developed after 1-2 months shown by significantly decreased ejection fraction (EF%) and deceleration time, respectively. These parameters together with the tricuspid annular plane systolic excursion (TAPSE) decreased again after 5-6 months. Soluble intercellular adhesion molecule-1 (sICAM-1) significantly increased in the heart homogenates at month 6, while other inflammatory cytokines were undetectable. This is the first study demonstrating smoking duration-dependent, complex cardiopulmonary alterations characteristic to COPD, in which inflammatory cytokine cascades and MMP-2/9 might be responsible for pulmonary destruction and sICAM-1 for heart dysfunction.
Assuntos
Doença Pulmonar Obstrutiva Crônica/patologia , Fumar/efeitos adversos , Poluição por Fumaça de Tabaco/estatística & dados numéricos , Animais , Líquido da Lavagem Broncoalveolar/química , Comorbidade , Modelos Animais de Doenças , Inflamação , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1alfa/metabolismo , Pulmão/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/patologia , Enfisema Pulmonar/fisiopatologia , Fumaça , NicotianaRESUMO
Transient Receptor Potential Ankyrin 1 (TRPA1) channels are localized on sensory nerves and several non-neural cells, but data on their functional significance are contradictory. We analysed the presence and alterations of TRPA1 in comparison with TRP Vanilloid 1 (TRPV1) at mRNA and protein levels in human and mouse intact and inflamed colons. The role of TRPA1 in a colitis model was investigated using gene-deficient mice. TRPA1 and TRPV1 expressions were investigated in human colon biopsies of healthy subjects and patients with inflammatory bowel diseases (IBD: ulcerative colitis, Crohn's disease) with quantitative PCR and immunohistochemistry. Mouse colitis was induced by oral 2% dextran-sulphate (DSS) for 10 days. For investigating the functions of TRPA1, Disease Activity Index (weight loss, stool consistency, blood content) was determined in C57BL/6-based Trpa1-deficient (knockout: KO) and wildtype (WT) mice. Sensory neuropeptides, their receptors, and inflammatory cytokines/chemokines were determined with qPCR or Luminex. In human and mouse colons TRPA1 and TRPV1 are located on epithelial cells, macrophages, enteric ganglia. Significant upregulation of TRPA1 mRNA was detected in inflamed samples. In Trpa1 KO mice, Disease Activity Index was significantly higher compared to WTs. It could be explained by the greater levels of substance P, neurokinins A and B, neurokinin 1 receptor, pituitary adenylate-cyclase activating polypeptide, vasoactive intestinal polypeptide, and also interleukin-1beta, macrophage chemoattractant protein-1, monokine induced by gamma interferon-1, tumor necrosis factor-alpha and B-lymphocyte chemoattractant in the distal colon. TRPA1 is upregulated in colitis and its activation exerts protective roles by decreasing the expressions of several proinflammatory neuropeptides, cytokines and chemokines.
Assuntos
Canais de Cálcio/fisiologia , Colite/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Canais de Potencial de Receptor Transitório/fisiologia , Regulação para Cima , Animais , Sequência de Bases , Canais de Cálcio/genética , Colite/metabolismo , Colo/metabolismo , Primers do DNA , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/metabolismo , Reação em Cadeia da Polimerase , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/genéticaRESUMO
OBJECTIVE AND DESIGN: The function of the neurokinin 1 (NK1) receptor was investigated in the DSS-induced mouse colitis model using NK1 receptor-deficient mice and the selective antagonist netupitant. SUBJECTS: Colitis was induced by oral administration of 20 mg/ml DSS solution for 7 days in C57BL/6 and Tacr1 KO animals (n = 5-7). TREATMENT: During the induction, one-half of the C57BL/6 and Tacr1 KO group received one daily dose of 6 mg/kg netupitant, administered intraperitoneally, the other half of the group received saline, respectively. METHODS: Disease activity index (DAI), on the basis of stool consistency, blood and weight loss, was determined over 7 days. Histological evaluation, myeloperoxidase (MPO) measurement, cytokine concentrations and receptor expression analysis were performed on the colon samples. RESULTS: NK1 receptors are up-regulated in the colon in response to DSS treatment. DSS increased DAI, histopathological scores, BLC, sICAM-1, IFN-γ, IL-16 and JE in wildtype mice, which were significantly reduced in NK1 receptor-deficient ones. NK1 receptor antagonism with netupitant significantly diminished DAI, inflammatory histopathological alterations, BLC, IFN-γ, IL-13 and IL-16 in wildtype mice, but not in the NK1-deficient ones. MPO was similarly elevated and netupitant significantly decreased its activity in both groups. CONCLUSIONS: NK1 receptor antagonism could be beneficial for colitis via inhibiting different inflammatory mechanisms.
Assuntos
Colite/tratamento farmacológico , Antagonistas dos Receptores de Neurocinina-1/uso terapêutico , Piridinas/uso terapêutico , Receptores da Neurocinina-1/fisiologia , Animais , Colite/induzido quimicamente , Colite/patologia , Colo/patologia , Citocinas/análise , Sulfato de Dextrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidase/metabolismo , Piridinas/farmacologia , Receptores da Neurocinina-1/genética , Índice de Gravidade de DoençaRESUMO
Alterations of somatostatin-like immunoreactivity (SST-LI) in the plasma of 11 systemic inflammatory response syndrome (SIRS) patients were investigated in correlation with cytokines, adhesion molecules and coagulation markers repeatedly during 4 days. The origin and role of SST were studied in the cecum ligation and puncture (CLP) rat SIRS model. Capsaicin-sensitive peptidergic sensory nerves were defunctionalized by resiniferatoxin (RTX) pretreatment 2 weeks earlier, in a separate group animals were treated with the somatostatin receptor antagonist cyclo-somatostatin (C-SOM). Plasma SST-LI significantly elevated in septic patients compared to healthy volunteers during the whole 4-day period. Significantly decreased Horowitz score showed severe lung injury, increased plasma C-reactive protein and procalcitonin confirmed SIRS. Soluble P-selectin, tissue plasminogen activator and the interleukin 8 and monocyte chemotactic protein-1 significantly increased, interleukin 6 and soluble CD40 ligand did not change, and soluble Vascular Adhesion Molecule-1 decreased. SST-LI significantly increased in rats both in the plasma and the lung 6h after CLP compared to sham-operation. After RTX pretreatment SST-LI was not altered in intact animals, but the SIRS-induced elevation was absent. Lung MPO activity significantly increased 6h following CLP compared to sham operation, which was significantly higher both after RTX-desensitization and C-SOM-treatment. Most non-pretreated operated rats survived the 6h, but 60% of the RTX-pretreated ones died showing a significantly worse survival. This is the first comprehensive study in humans and animal experiments providing evidence that SST is released from the activated peptidergic sensory nerves. It gets into the bloodstream and mediates a potent endogenous protective mechanism.
Assuntos
Peptídeos/sangue , Sepse/sangue , Síndrome de Resposta Inflamatória Sistêmica/sangue , Idoso , Animais , Biomarcadores/sangue , Ligante de CD40/sangue , Capsaicina/farmacologia , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Peptídeos/imunologia , Ratos Wistar , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Sepse/imunologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Molécula 1 de Adesão de Célula Vascular/sangueRESUMO
Tachykinins encoded by the preprotachykinin A (TAC1) gene such as substance P (SP) and neurokinin A (NKA) are involved in neurogenic inflammatory processes via predominantly neurokinins 1 and 2 (NK1 and NK2) receptor activation, respectively. Endokinins and hemokinins encoded by the TAC4 gene also have remarkable selectivity and potency for the NK1 receptors and might participate in inflammatory cell functions. The aim of the present study was to investigate endotoxin-induced airway inflammation and consequent bronchial hyper-reactivity in TAC1(-/-), NK1(-/-) and also in double knockout (TAC1(-/-)/NK1(-/-)) mice. Sub-acute interstitial lung inflammation was evoked by intranasal Escherichia coli lipopolysaccharide (LPS) in the knockout mice and their wildtype C57BL/6 counterparts 24 h before measurement. Respiratory parameters were measured with unrestrained whole body plethysmography. Bronchoconstriction was induced by inhalation of the muscarinic receptor agonist carbachol and Penh (enhanced pause) correlating with airway resistance was calculated. Lung interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) concentrations were measured with ELISA. Histological evaluation was performed and a composite morphological score was determined. Myeloperoxidase (MPO) activity in the lung was measured with spectrophotometry to quantify the number of infiltrating neutrophils/macrophages. Airway hyper-reactivity was significantly reduced in the TAC1(-/-) as well as the TAC1(-/-)/NK1(-/-) groups. However, LPS-induced histological inflammatory changes (perivascular/peribronchial oedema, neutrophil infiltration and goblet cell hyperplasia), MPO activity and TNF-alpha concentration were markedly diminished only in TAC1(-/-) mice. Interestingly, the concentrations of both cytokines, IL-1beta and TNF-alpha, were significantly greater in the NK1(-/-) group. These data clearly demonstrated on the basis of histology, MPO and cytokine measurements that TAC1 gene-derived tachykinins, SP and NKA, play a significant role in the development of endotoxin-induced murine airway inflammation, but not solely via NK1 receptor activation. However, in inflammatory bronchial hyper-responsiveness other tachykinins, such as hemokinin-1 acting through NK1 receptors also might be involved.
Assuntos
Pulmão/metabolismo , Pneumonia/metabolismo , Precursores de Proteínas/metabolismo , Receptores da Neurocinina-1/metabolismo , Taquicininas/metabolismo , Análise de Variância , Animais , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Broncoconstrição/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Peroxidase/metabolismo , Pletismografia Total , Pneumonia/induzido quimicamente , Espectrofotometria , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The aim of this study was to investigate the involvement of transient receptor potential vanilloid 1 (TRPV1) receptors in oral dextran sulfate sodium-induced (DSS) colitis using TRPV1 knockout mice and their wild-type C57BL/6 counterparts. DSS (2% or 5%) was administered orally ad libitum for 7 days; the controls received tap water. Animal weight, stool consistency, and blood content were scored every day to calculate the disease activity index (DAI). After sacrificing the mice on day 7, the colons were cut into three equal segments (proximal, intermediate, and distal) for histology, myeloperoxidase (MPO), and cytokine measurements. In the 2% DSS-treated group, the lack of TRPV1 receptors decreased the DAI. Each colon segment of wild-type animals showed more than two-fold increase of MPO activity and more severe histological changes compared to the knockouts. This difference was not observed in case of 5% DSS, when extremely severe inflammation occurred in both groups. IL-1beta production was not altered by the absence of TRPV1. In conclusion, activation of TRPV1 channels enhances the clinical symptoms, histopathological changes, and neutrophil accumulation induced by 2% DSS. Elucidating the modulator role of TRPV1 channels in inflammatory bowel diseases may contribute to the development of novel anti-inflammatory drugs for their therapy.