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BACKGROUND: The inflammation in the lungs and other vital organs in COVID-19 are characterized by the presence of neutrophils and high concentration of neutrophil extracellular traps (NETs), which also seems to mediate host tissue damage. However, it is not known whether NETs could have virucidal activity against SARS-CoV-2. METHODS: We investigated whether NETs could prevent SARS-CoV-2 replication in neutrophils and epithelial cells, and what the consequence of NETs degradation in K18-humanized ACE2 transgenic mice infected with SARS-CoV-2. RESULTS: Here, by immunofluorescence microscopy we observed that viral particles co-localize with NETs in neutrophils isolated from COVID-19 patients or from healthy individuals and infected in vitro. The inhibition of NETs production increased virus replication in neutrophils. In parallel, we observed that NETs inhibited virus abilities to infect and replicate in epithelial cells after 24â h of infection. Degradation of NETs with DNase I prevented their virucidal effect in vitro. Using K18-humanized ACE2 transgenic mice we observed a higher viral load in animals treated with DNase I. On the other hand, the virucidal effect of NETs was not dependent on neutrophil elastase or myeloperoxidase activity. CONCLUSION: Our results provide evidence of the role of NETosis as a mechanism of SARS-CoV-2 viral capture and inhibition.
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Multiple organ dysfunction is the most severe outcome of sepsis progression and is highly correlated with a worse prognosis. Excessive neutrophil extracellular traps (NETs) are critical players in the development of organ failure during sepsis. Therefore, interventions targeting NET release would likely effectively prevent NET-based organ injury associated with this disease. Herein, we demonstrate that the pore-forming protein gasdermin D (GSDMD) is active in neutrophils from septic humans and mice and plays a crucial role in NET release. Inhibition of GSDMD with disulfiram or genic deletion abrogated NET formation, reducing multiple organ dysfunction and sepsis lethality. Mechanistically, we demonstrate that during sepsis, activation of the caspase-11/GSDMD pathway controls NET release by neutrophils during sepsis. In summary, our findings uncover a novel therapeutic use for disulfiram and suggest that GSDMD is a therapeutic target to improve sepsis treatment.
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Trampas Extracelulares/genética , Eliminación de Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Insuficiencia Multiorgánica/genética , Proteínas de Unión a Fosfato/genética , Sepsis/genética , Inhibidores del Acetaldehído Deshidrogenasa/uso terapéutico , Traslado Adoptivo , Anciano , Animales , Células Cultivadas , Disulfiram/uso terapéutico , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Insuficiencia Multiorgánica/patología , Insuficiencia Multiorgánica/terapia , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Sepsis/patología , Sepsis/terapiaRESUMEN
BACKGROUND: COVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. METHODS: Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. SARS-CoV-2-infected K18-hACE2 mice were performed for clinical sickness scores and lung pathology. Moreover, the levels of NETs were assessed and lung injuries were by histopathology and TUNEL assay. Finally, the injury in the heart and kidney was assessed by histopathology and biochemical-specific markers. RESULTS: DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potentially deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. CONCLUSIONS: Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.
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Lesión Pulmonar Aguda , COVID-19 , Trampas Extracelulares , Animales , Humanos , Ratones , SARS-CoV-2 , Tratamiento Farmacológico de COVID-19 , Modelos Animales de Enfermedad , Neutrófilos , Desoxirribonucleasa I/farmacología , Desoxirribonucleasa I/uso terapéuticoRESUMEN
OBJECTIVE: This study aimed to investigate the effects of FK506 on experimental sepsis immunopathology. It investigated the effect of FK506 on leukocyte recruitment to the site of infection, systemic cytokine production, and organ injury in mice with sepsis. METHODS: Using a murine cecal ligation and puncture (CLP) peritonitis model, the experiments were performed with wild-type (WT) mice and mice deficient in the gene Nfat1 (Nfat1-/-) in the C57BL/6 background. Animals were treated with 2.0 mg/kg of FK506, subcutaneously, 1 h before the sepsis model, twice a day (12 h/12 h). The number of bacteria colony forming units (CFU) was manually counted. The number of neutrophils in the lungs was estimated by the myeloperoxidase (MPO) assay. The expression of CXCR2 in neutrophils was determined using flow cytometry analysis. The expression of inflammatory cytokines in macrophage was determined using ELISA. The direct effect of FK506 on CXCR2 internalization was evaluated using HEK-293T cells after CXCL2 stimulation by the BRET method. RESULTS: FK506 treatment potentiated the failure of neutrophil migration into the peritoneal cavity, resulting in bacteremia and an exacerbated systemic inflammatory response, which led to higher organ damage and mortality rates. Failed neutrophil migration was associated with elevated CXCL2 chemokine plasma levels and lower expression of the CXCR2 receptor on circulating neutrophils compared with non-treated CLP-induced septic mice. FK506 did not directly affect CXCL2-induced CXCR2 internalization by transfected HEK-293 cells or mice neutrophils, despite increasing CXCL2 release by LPS-treated macrophages. Finally, the CLP-induced response of Nfat1-/- mice was similar to those observed in the Nfat1+/+ genotype, suggesting that the FK506 effect is not dependent on the NFAT1 pathway. CONCLUSION: Our data indicate that the increased susceptibility to infection of FK506-treated mice is associated with failed neutrophil migration due to the reduced membrane availability of CXCR2 receptors in response to exacerbated levels of circulating CXCL2.
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Neutrófilos , Sepsis , Humanos , Ratones , Animales , Tacrolimus/farmacología , Tacrolimus/uso terapéutico , Células HEK293 , Ratones Endogámicos C57BL , Sepsis/metabolismo , Infiltración NeutrófilaRESUMEN
BACKGROUND: Although the literature shows that an increase in both the number and suppressive function of CD4+forkhead box P3 (FOXP3)+ T-regulatory cells (Tregs) during sepsis contributes to an immunosuppressed state, little is known about the identity of these cells. METHODS: Using the sepsis mouse model of cecal ligation and puncture (CLP), we analyzed the frequency and molecular signature of the T-cell immunoglobulin and ITIM domain (TIGIT)+ and TIGIT- Treg subsets, using flow cytometry and quantitative polymerase chain reaction. In addition, ST2-/- and signal transducer and activator of transcription 6 (STAT6)-/- mice were submitted to CLP or recombinant interleukin 33 (IL-33) treatment to investigate the mechanism whereby TIGIT+ Tregs differentiate during sepsis. RESULTS: Sepsis was marked by the sustained expansion of the highly suppressive TIGIT+ Treg subset, which expresses Helios, neuropilin 1, and high levels of Tnfrsf18 and Pdcd1 at 15 days after CLP. The increase in TIGIT+ Tregs was accompanied by higher susceptibility to nosocomial bacteria challenge, suggesting their association with post sepsis immunosuppression. Mechanistically, we found that the ST2 deletion abrogated the expansion of the TIGIT+ Treg subset during sepsis. Furthermore, treatment with recombinant IL-33 resulted in the expansion of TIGIT+ Tregs depending on the STAT6 and M2 macrophages. CONCLUSIONS: These findings demonstrated that only the TIGIT+ Tregs remain stably expanded at the late phase of sepsis. Moreover, the expansion of TIGIT+ Tregs is dependent on the IL-33/ST2/STAT6/M2 macrophage axis.
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Sepsis , Linfocitos T Reguladores , Animales , Factores de Transcripción Forkhead/genética , Terapia de Inmunosupresión , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Ratones , Receptores Inmunológicos/genéticaRESUMEN
BACKGROUND: The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. OBJECTIVES: We aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19. METHODS: We performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection. RESULTS: We found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage. CONCLUSION: These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy.
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Tratamiento Farmacológico de COVID-19 , Trampas Extracelulares , Animales , Disulfiram/metabolismo , Trampas Extracelulares/metabolismo , Ratones , Neutrófilos/metabolismo , SARS-CoV-2RESUMEN
Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.
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Neutrófilos , Piruvato Quinasa , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neutrófilos/metabolismo , Fosforilación , GlucólisisRESUMEN
The TIGIT+FOXP3+Treg subset (TIGIT+Tregs) exerts robust suppressive activity on cellular immunity and predisposes septic individuals to opportunistic infection. We hypothesized that TIGIT+Tregs could play an important role in intensifying the COVID-19 severity and hampering the defense against nosocomial infections during hospitalization. Herein we aimed to verify the association between the levels of the TIGIT+Tregs with the mechanical ventilation requirement, fatal outcome, and bacteremia during hospitalization. TIGIT+Tregs were immunophenotyped by flow cytometry from the peripheral blood of 72 unvaccinated hospitalized COVID-19 patients at admission from May 29th to August 6th, 2020. The patients were stratified during hospitalization according to their mechanical ventilation requirement and fatal outcome. COVID-19 resulted in a high prevalence of the TIGIT+Tregs at admission, which progressively increased in patients with mechanical ventilation needs and fatal outcomes. The prevalence of TIGIT+Tregs positively correlated with poor pulmonary function and higher plasma levels of LDH, HMGB1, FGL2, and TNF. The non-survivors presented higher plasma levels of IL-33, HMGB1, FGL2, IL-10, IL-6, and 5.54 times more bacteremia than survivors. Conclusions: The expansion of the TIGIT+Tregs in COVID-19 patients was associated with inflammation, lung dysfunction, bacteremia, and fatal outcome.
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Bacteriemia , COVID-19 , Infección Hospitalaria , Proteína HMGB1 , Humanos , Respiración Artificial , Linfocitos T Reguladores , Receptores Inmunológicos , FibrinógenoRESUMEN
Sepsis is a lethal syndrome characterized by systemic inflammation and abnormal coagulation. Despite therapeutic advances, sepsis mortality remains substantially high. Herein, we investigated the role of the plasminogen/plasmin (Plg/Pla) system during sepsis. Plasma levels of Plg were significantly lower in mice subjected to severe compared with nonsevere sepsis, whereas systemic levels of IL-6, a marker of sepsis severity, were higher in severe sepsis. Plg levels correlated negatively with IL-6 in both septic mice and patients, whereas plasminogen activator inhibitor-1 levels correlated positively with IL-6. Plg deficiency render mice susceptible to nonsevere sepsis induced by cecal ligation and puncture (CLP), resulting in greater numbers of neutrophils and M1 macrophages, liver fibrin(ogen) deposition, lower efferocytosis, and increased IL-6 and neutrophil extracellular trap (NET) release associated with organ damage. Conversely, inflammatory features, fibrin(ogen), and organ damage were substantially reduced, and efferocytosis was increased by exogenous Pla given during CLP- and LPS-induced endotoxemia. Plg or Pla protected mice from sepsis-induced lethality and enhanced the protective effect of antibiotics. Mechanistically, Plg/Pla-afforded protection was associated with regulation of NET release, requiring Pla-protease activity and lysine binding sites. Plg/Pla are important host-protective players during sepsis, controlling local and systemic inflammation and collateral organ damage.
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Trampas Extracelulares , Sepsis , Ratones , Animales , Fibrinolisina , Plasminógeno , Trampas Extracelulares/metabolismo , Interleucina-6/metabolismo , Inflamación/metabolismo , Sepsis/metabolismo , Fibrina/metabolismoRESUMEN
Cytotoxic agents synergize with immune checkpoint inhibitors and improve outcomes for patients with several cancer types. Nonetheless, a parallel increase in the incidence of dose-limiting side effects, such as peripheral neuropathy, is often observed. Here, we investigated the role of the programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis in the modulation of paclitaxel-induced neuropathic pain. We found that human and mouse neural tissues, including the dorsal root ganglion (DRG), expressed basal levels of PD-1 and PD-L1. During the development of paclitaxel-induced neuropathy, an increase in PD-L1 expression was observed in macrophages from the DRG. This effect depended on Toll-like receptor 4 activation by paclitaxel. Furthermore, PD-L1 inhibited pain behavior triggered by paclitaxel or formalin in mice, suggesting that PD-1/PD-L1 signaling attenuates peripheral neuropathy development. Consistent with this, we observed that the combined use of anti-PD-L1 plus paclitaxel increased mechanical allodynia and chronic neuropathy development induced by single agents. This effect was associated with higher expression of inflammatory markers (Tnf, Il6, and Cx3cr1) in peripheral nervous tissue. Together, these results suggest that PD-1/PD-L1 inhibitors enhance paclitaxel-induced neuropathic pain by suppressing PD-1/PD-L1 antinociceptive signaling.
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Antineoplásicos Fitogénicos , Neuralgia , Ratas , Humanos , Ratones , Animales , Receptor de Muerte Celular Programada 1 , Antineoplásicos Fitogénicos/efectos adversos , Ratas Sprague-Dawley , Neuralgia/inducido químicamente , Neuralgia/metabolismo , Paclitaxel , Analgésicos/efectos adversosRESUMEN
Vigorous exercise can induce gastrointestinal disorders such decreased gastric emptying pace, while low-intensity exercise can accelerate gastric motility. However, the mechanisms of these effects are still unknown. We investigated the possible neurohumoral mechanisms involved in these phenomena. In sedentary (Sed) and acute exercise (Ex) groups of rats, we assessed the activation of c-Fos in NTS and DVMN and the plasma levels of CCK and OXT. Separate groups received pretreatment with the oxytocin antagonist atosiban (AT), the cholecystokinin antagonist devazepide (DVZ), or the TRPV1 receptor inhibitor capsazepine (CAPZ). AT, DVZ and CAPZ treatments prevented (p<0.05) slower gastric emptying induced by acute exercise. The gene expression of OXT decreased (P<0.05) while that of CCK increased (P<0.05) in the gastric fundus and pylorus of the Ex group, while the plasma levels of OXT rose (p<0.05) and of CCK declined (p<5.05). We also observed activation (p<0.05) of c-Fos-sensitive neurons in the NTS and DVMN of exercised rats. In conclusion, acute exercise slowed gastric emptying by the vagal afferent pathway, which involved activation of CCK1/OXT/TRPV1 sensitivity.
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Colecistoquinina , Vaciamiento Gástrico , Animales , Antagonistas de Hormonas/farmacología , Oxitocina , Ratas , Nervio VagoRESUMEN
Colorectal cancer (CRC) is a disease with high incidence and mortality. Colonoscopy is a gold standard among tests used for CRC traceability. However, serious complications, such as colon perforation, may occur. Non-invasive diagnostic procedures are an unmet need. We aimed to identify a plasma microRNA (miRNA) signature for CRC detection. Plasma samples were obtained from subjects (n = 109) at different stages of colorectal carcinogenesis. The patients were stratified into a non-cancer (27 healthy volunteers, 17 patients with hyperplastic polyps, 24 with adenomas), and a cancer group (20 CRC and 21 metastatic CRC). miRNAs (381) were screened by TaqMan Low-Density Array. A classifier based on four differentially expressed miRNAs (miR-28-3p, let-7e-5p, miR-106a-5p, and miR-542-5p) was able to discriminate cancer versus non-cancer cases. The overexpression of these miRNAs was confirmed by RT-qPCR, and a cross-study validation step was implemented using eight data series retrieved from Gene Expression Omnibus (GEO). In addition, another external data validation using CRC surgical specimens from The Cancer Genome Atlas (TCGA) was carried out. The predictive model's performance in the validation set was 76.5% accuracy, 59.4% sensitivity, and 86.8% specificity (area under the curve, AUC = 0.716). The employment of our model in the independent publicly available datasets confirmed a good discrimination performance in five of eight datasets (median AUC = 0.823). Applying this algorithm to the TCGA cohort, we found 99.5% accuracy, 99.7% sensitivity, and 90.9% specificity (AUC = 0.998) when the model was applied to solid colorectal tissues. Overall, we suggest a novel signature of four circulating miRNAs, i.e., miR-28-3p, let-7e-5p, miR-106a-5p, and miR-542-5p, as a predictive tool for the detection of CRC.
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The inflammasomes control the bioactivity of pro-inflammatory cytokines of the interleukin (IL)-1 family. The inflammasome assembled by NLRP3 has been predominantly studied in homogeneous cell populations in vitro, neglecting the influence of cellular interactions that occur in vivo. Here, we show that platelets boost the inflammasome capacity of human macrophages and neutrophils and are critical for IL-1 production by monocytes. Platelets license NLRP3 transcription, thereby enhancing ASC oligomerization, caspase-1 activity, and IL-1ß secretion. Platelets influence IL-1ß production in vivo, and blood platelet counts correlate with plasmatic IL-1ß levels in malaria. Furthermore, we reveal an enriched platelet gene signature among the highest-expressed transcripts in IL-1ß-driven autoinflammatory diseases. The platelet effect is independent of cell-to-cell contact, platelet-derived lipid mediators, purines, nucleic acids, and a host of platelet cytokines, and it involves the triggering of calcium-sensing receptors on macrophages. Hence, platelets provide an additional layer of regulation of inflammasomes and IL-1-driven inflammation.
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Plaquetas/inmunología , Inmunidad Innata/inmunología , Inflamasomas/metabolismo , Interleucina-1beta/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , HumanosRESUMEN
BACKGROUND: Previous data have reported that the growth of established tumors may be facilitated by postsepsis disorder through changes in the microenvironment and immune dysfunction. However, the influence of postsepsis disorder in initial carcinogenesis remains elusive. METHODS: In the present work, the effect of postsepsis on inflammation-induced early carcinogenesis was evaluated in an experimental model of colitis-associated colorectal cancer (CAC). We also analyzed the frequency and role of intestinal T regulatory cells (Treg) in CAC carcinogenesis. RESULTS: The colitis grade and the tumor development rate were evaluated postmortem or in vivo through serial colonoscopies. Sepsis-surviving mice (SSM) presented with a lower colonic DNA damage, polyp incidence, reduced tumor load, and milder colitis than their sham-operated counterparts. Ablating Treg led to restoration of the ability to develop colitis and tumor polyps in the SSM, in a similar fashion to that in the sham-operated mice. On the other hand, the growth of subcutaneously inoculated MC38luc colorectal cancer cells or previously established chemical CAC tumors was increased in SSM. CONCLUSION: Our results provide evidence that postsepsis disorder has a dual effect in cancer development, inhibiting inflammation-induced early carcinogenesis in a Treg-dependent manner, while increasing the growth of previously established tumors.
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Colitis/complicaciones , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Inflamación/complicaciones , Sepsis/complicaciones , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología , Animales , Colitis/inmunología , Colitis/patología , Neoplasias del Colon/etiología , Citocinas/metabolismo , Femenino , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Sepsis/inmunología , Sepsis/patología , Transducción de SeñalRESUMEN
Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, we investigated whether NETs would be involved in COVID-19 pathophysiology. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and healthy controls were enrolled. The concentration of NETs was augmented in plasma, tracheal aspirate, and lung autopsies tissues from COVID-19 patients, and their neutrophils released higher levels of NETs. Notably, we found that viable SARS-CoV-2 can directly induce the release of NETs by healthy neutrophils. Mechanistically, NETs triggered by SARS-CoV-2 depend on angiotensin-converting enzyme 2, serine protease, virus replication, and PAD-4. Finally, NETs released by SARS-CoV-2-activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represents a potential therapeutic target for COVID-19.
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Betacoronavirus/fisiología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Trampas Extracelulares/fisiología , Neumonía Viral/inmunología , Neumonía Viral/virología , Células A549 , Adulto , Enzima Convertidora de Angiotensina 2 , COVID-19 , Muerte Celular , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/patología , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Células HeLa , Humanos , Masculino , Activación Neutrófila , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/sangre , Neumonía Viral/patología , SARS-CoV-2 , Serina Proteasas/metabolismo , Succión , Tráquea/inmunologíaRESUMEN
Ifosfamide (IFO) is an antineoplastic drug that is commonly used to treat gynecological and breast cancers. Hemorrhagic cystitis (HC) is a common side effect associated with IFO injection, which courses with neutrophil accumulation and affects 6-50% of patients depending on dose intensity. Here, we investigated the role of neutrophils in this inflammatory process. Female Swiss mice (nâ¯=â¯8/group) were injected with saline, IFO (400â¯mg/kg, i.p.), fucoidan (a P- and L-selectins inhibitor, 100â¯mg/kg, i.v.) or IFOâ¯+â¯fucoidan (1-100â¯mg/kg) alone or combined with mesna (80â¯mg/kg i.p.). Another group of mice received anti-Ly6G antibody (500⯵g/mouse, once daily for 2â¯days) for neutrophil depletion before IFO injection. In another experimental setting, animals received granulocyte colony-stimulating factor (G-CSF, 400⯵g/kg), IFO (200â¯mg/kg), G-CSF (25-400⯵g/kg, for 5â¯days)â¯+â¯IFO (200â¯mg/kg, i.p.) or fucoidanâ¯+â¯G-CSFâ¯+â¯IFO. Bladder injury was evaluated 12â¯h after IFO injection. IFO 400â¯mg/kg significantly increased visceral hyperalgesia, bladder edema, hemorrhage, vascular permeability, MPO, IL-1ß and IL-6 tissue levels, and COX-2 immunostaining and expression versus the saline group (Pâ¯<â¯0.05). Conversely, fucoidan (100â¯mg/kg) significantly attenuated these parameters compared to IFO-injected mice (Pâ¯<â¯0.05). Additionally, fucoidan potentiated mesna protective effect when compared with IFOâ¯+â¯mesna group (Pâ¯<â¯0.05). Accordingly, neutrophil depletion with anti-Ly6G reduced inflammatory parameters and bladder injury compared to IFO (Pâ¯<â¯0.05). In contrast, G-CSF enhanced IFO (200â¯mg/kg)-induced HC, which was significantly attenuated by treatment with fucoidan (Pâ¯<â¯0.05). Therefore, neutrophils contribute to the pathogenesis of HC.
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Antineoplásicos Alquilantes/efectos adversos , Cistitis/inducido químicamente , Hemorragia/inducido químicamente , Ifosfamida/efectos adversos , Infiltración Neutrófila/efectos de los fármacos , Sustancias Protectoras/uso terapéutico , Animales , Cistitis/inmunología , Cistitis/patología , Cistitis/prevención & control , Quimioterapia Combinada , Femenino , Hemorragia/inmunología , Hemorragia/patología , Hemorragia/prevención & control , Mesna/administración & dosificación , Mesna/uso terapéutico , Ratones , Polisacáridos/administración & dosificación , Polisacáridos/uso terapéutico , Sustancias Protectoras/administración & dosificaciónRESUMEN
ß-Citronellol is a monoterpene found in the essential oil of various plants with antihypertensive properties. In fact, ß-citronellol possesses hypotensive actions due to its vasodilator abilities. Here we aimed to show that ß-citronellol recruits airway sensory neural circuitry to evoke cardiorespiratory effects. In anesthetized rats, intravenous injection of ß-citronellol caused biphasic hypotension, bradycardia and apnea. Bilateral vagotomy, perivagal capsaicin treatment or injection into the left ventricle abolished first rapid phase (named P1) but not delayed phase P2 of the ß-citronellol effects. P1 persisted after pretreatment with capsazepine, ondansetron, HC-030031 or suramin. Suramin abolished P2 of apnea. In awake rats, ß-citronellol induced biphasic hypotension and bradycardia being P1 abolished by methylatropine. In vitro, ß-citronellol inhibited spontaneous or electrically-evoked contractions of rat isolated right or left atrium, respectively, and fully relaxed sustained contractions of phenylephrine in mesenteric artery rings. In conclusion, chemosensitive pulmonary vagal afferent fibers appear to mediate the cardiovascular and respiratory effects of ß-citronellol. The transduction mechanism in P1 seems not to involve the activation of transient receptor potential vanilloid subtype 1 (TRPV1), transient receptor potential ankyrin subtype 1 (TRPA1), purinergic (P2X) or 5-HT3 receptors located on airways sensory nerves. P2 of hypotension and bradycardia seems resulting from a cardioinhibitory and vasodilatory effect of ß-citronellol and the apnea from a purinergic signaling.
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Apnea/inducido químicamente , Bradicardia/inducido químicamente , Hipotensión/inducido químicamente , Monoterpenos/farmacología , Monoterpenos Acíclicos , Animales , Apnea/fisiopatología , Presión Arterial/efectos de los fármacos , Función Atrial/efectos de los fármacos , Bradicardia/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Hipotensión/fisiopatología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Ratas Wistar , Frecuencia Respiratoria/efectos de los fármacos , Vagotomía , Nervio Vago/efectos de los fármacos , Nervio Vago/fisiologíaRESUMEN
Homeostasis of blood volume (BV) is attained through a functional interaction between the cardiovascular and renal systems. The gastrointestinal tract also adjusts its permeability and motor behavior after acute BV imbalances. We evaluated the effect of progressive nephron loss on gut motility. Male Wistar rats were subjected or not (sham) to 5/6 partial nephrectomy (PNX) in two steps (0 and 7th day). After further 3, 7, or 14 days, PNX and sham operation (control) rats were instrumented to monitor mean arterial pressure (MAP), central venous pressure (CVP), heart rate (HR), and blood collection for biochemical analysis. The next day, they were gavage fed with a liquid test meal (phenol red in glucose solution), and fractional dye recovery determined 10, 20, or 30 min later. The effect of nonhypotensive hypovolemia and the role of neuroautonomic pathways on PNX-induced gastric emptying (GE) delay were also evaluated. Compared with the sham-operated group, PNX rats exhibited higher (P < 0.05) MAP and CVP values as well as increased values of gastric dye recovery, phenomenon proportional to the BV values. Gastric retention was prevented by prior hypovolemia, bilateral subdiaphragmatic vagotomy, coelic ganglionectomy + splanchnicectomy, guanethidine, or atropine pretreatment. PNX also inhibited (P < 0.05) the marker's progression through the small intestine. In anesthetized rats, PNX increased (P < 0.05) gastric volume, measured by a balloon catheter in a barostat system. In conclusion, the progressive loss of kidney function delayed the GE rate, which may contribute to gut dysmotility complaints associated with severe renal failure.
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OBJECTIVE: To develop an experimental model of stable saccular aneurysm in carotid of pigs using the internal jugular vein. METHODS: In 12 healthy pigs, weighing between 25 and 5 0kg, five males and seven females, we made a right common carotid artery aneurysm. After elliptical arteriotomy, we carried out a terminolateral anastomosis with the distal stump of the internal jugular vein. Aneurysm volume was calculated so that the value did not exceed 27 times the area of the arteriotomy. After six days angiography and microscopic examination were performed to assess patency of the aneurysm and the presence of total or partial thrombosis. RESULTS: There was a significant weight gain of pigs in the time interval between the manufacture of the aneurysm and angiography (p = 0.04). Aneurysmal patency was observed in ten pigs (83%). Operative wound infections occurred in two animals (16.6%), both with early onset, three days after the making of the aneurysm. Histological analysis showed aneurysm thrombus partially occluding the light in nine pigs (75%). In these animals, it was observed that on average 9% of the aneurysmal diameter was filled with thrombi. CONCLUSION: It was possible to develop a stable experimental model of saccular aneurysms in pig carotid artery by use of the internal jugular vein.
Asunto(s)
Aneurisma , Enfermedades de las Arterias Carótidas , Modelos Animales de Enfermedad , Venas Yugulares , Animales , Femenino , Masculino , PorcinosRESUMEN
OBJETIVO: Desenvolver um modelo experimental estável de aneurisma sacular em carótida de suínos utilizando veia jugular interna. MÉTODOS: Em 12 suínos sadios, com peso variando entre 25 e 50kg, cinco machos e sete fêmeas, foi confeccionado aneurisma na artéria carótida comum direita. Após arteriotomia elíptica, foi realizada anastomose terminolateral com coto distal de veia jugular interna. O volume do aneurisma era calculado de maneira que o valor não excedesse em 27 vezes o valor da área da arteriotomia. Após seis dias, era realizada angiografia e análise microscópica do aneurisma para avaliar perviedade e trombose parcial ou total. RESULTADOS: Houve ganho de peso significante dos suínos no intervalo de tempo entre a confecção do aneurisma e a angiografia (p = 0,04). Foi observada perviedade aneurismática em dez suínos (83%). Ocorreram infecções de feridas operatórias em dois animais (16,6%), ambas com início de aparecimento em três dias após a confecção do aneurisma. Análise histológica dos aneurismas mostrou trombos ocluindo parcialmente a luz em nove suínos (75%). Nesses animais, observou-se que, em média, 9% da luz aneurismática estava preenchida por trombos. CONCLUSÃO: Pôde ser desenvolvido um modelo experimental estável de aneurisma sacular em carótida de suínos utilizando veia jugular interna.
OBJECTIVE: To develop an experimental model of stable saccular aneurysm in carotid of pigs using the internal jugular vein. METHODS: In 12 healthy pigs, weighing between 25 and 50kg, five males and seven females, we made a right common carotid artery aneurysm. After elliptical arteriotomy, we carried out a terminolateral anastomosis with the distal stump of the internal jugular vein. Aneurysm volume was calculated so that the value did not exceed 27 times the area of the arteriotomy. After six days angiography and microscopic examination were performed to assess patency of the aneurysm and the presence of total or partial thrombosis. RESULTS: There was a significant weight gain of pigs in the time interval between the manufacture of the aneurysm and angiography (p = 0.04). Aneurysmal patency was observed in ten pigs (83%). Operative wound infections occurred in two animals (16.6%), both with early onset, three days after the making of the aneurysm. Histological analysis showed aneurysm thrombus partially occluding the light in nine pigs (75%). In these animals, it was observed that on average 9% of the aneurysmal diameter was filled with thrombi. CONCLUSION: It was possible to develop a stable experimental model of saccular aneurysms in pig carotid artery by use of the internal jugular vein.