RESUMO
OBJECTIVE: This study aims to examine the effect of sepsis on the dynamics of skeletal muscle partial oxygen pressure during muscle contractions as well as the effect of reactive oxygen species (ROS) scavenger (ascorbic acid, Asc). METHODS: Twenty-seven male Sprague-Dawley rats (2-3 months old) were randomly assigned to three groups; sham, cecal ligation and puncture (CLP), or CLP plus ascorbic acid treatment group (CLP + Asc). Electrical stimuli-induced muscle contractions and partial oxygen pressure measurements were performed at 3 h after CLP. The interstitial oxygen pressure (PO2 is) in the spinotrapezius muscle was measured by the phosphorescence quenching method. RESULTS: The PO2 is at rest was not different between the three groups. The PO2 is decreased from rest to contraction in all groups. Compared to the sham, the time to decrease PO2 is was significantly faster in CLP but not in CLP + Asc (p < .05). Compared to the sham, the PO2 is during muscle contractions was significantly lower in both CLP and CLP + Asc (p < .05, respectively). CONCLUSIONS: Our results suggest that CLP-induced sepsis accelerated the decay of PO2 is at the onset of muscle contractions and maintained a low level of PO2 is during muscle contractions.
Assuntos
Espécies Reativas de Oxigênio , Sepse , Animais , Masculino , Ratos , Ácido Ascórbico/farmacologia , Músculo Esquelético/fisiologia , Oxigênio , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/farmacologiaRESUMO
OBJECTIVE: This study aimed to clarify the effect of Type I diabetes (DIA) on transcapillary PO2 gradients, which are oxygen-driving factors between the blood and the interstitium, in the contracting muscle of rats. METHODS: Wistar male rats were divided into the diabetic (streptozocin i.p.) and sham groups. Microvascular and interstitial PO2 were measured in the extensor digitorum longus muscle during electrical stimulation-induced muscle contraction, using the phosphorescence quenching method. Transcapillary PO2 gradient, ΔPO2, was calculated as microvascular minus interstitial PO2. RESULTS: Resting microvascular PO2 was higher in the diabetic group than in the sham group (6.3 ± 1.7 vs. 4.7 ± 0.9 mmHg, p < 0.05) and remained for 180 s. Interstitial PO2 from rest to muscle contraction did not differ between the groups. The ΔPO2 was higher in the diabetic group than in the sham group at rest and during muscle contraction (4.03 ± 1.42 vs. 2.46 ± 0.90 mmHg at rest; 3.67 ± 1.51 vs. 2.22 ± 0.65 mmHg during muscle contraction, p < 0.05). Marked muscle atrophy was observed in the diabetic group. CONCLUSION: DIA increased microvascular and transcapillary PO2 gradients in the skeletal muscle. The enhanced PO2 gradients were maintained from rest to muscle contraction in diabetic muscle.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Contração Muscular , Músculo Esquelético , Oxigênio , Ratos Wistar , Animais , Masculino , Ratos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Músculo Esquelético/irrigação sanguínea , Oxigênio/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 1/metabolismo , Capilares/metabolismo , Capilares/fisiopatologia , Capilares/patologia , Microcirculação , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologia , Atrofia Muscular/patologiaRESUMO
A high salt intake exacerbates insulin resistance, evoking hypertension due to systemic perivascular inflammation, oxidative-nitrosative stress and endothelial dysfunction. Angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blockers (ARBs) have been shown to abolish inflammation and redox stress but only partially restore endothelial function in mesenteric vessels. We investigated whether sympatho-adrenal overactivation evokes coronary vascular dysfunction when a high salt intake is combined with insulin resistance in male Goto-Kakizaki (GK) and Wistar rats treated with two different classes of ß-blocker or vehicle, utilising synchrotron-based microangiography in vivo. Further, we examined if chronic carvedilol (CAR) treatment preserves nitric oxide (NO)-mediated coronary dilation more than metoprolol (MET). A high salt diet (6% NaCl w/w) exacerbated coronary microvessel endothelial dysfunction and NO-resistance in vehicle-treated GK rats while Wistar rats showed modest impairment. Microvascular dysfunction was associated with elevated expression of myocardial endothelin, inducible NO synthase (NOS) protein and 3-nitrotyrosine (3-NT). Both CAR and MET reduced basal coronary perfusion but restored microvessel endothelium-dependent and -independent dilation indicating a role for sympatho-adrenal overactivation in vehicle-treated rats. While MET treatment reduced myocardial nitrates, only MET treatment completely restored microvessel dilation to dobutamine (DOB) stimulation in the absence of NO and prostanoids (combined inhibition), indicating that MET restored the coronary flow reserve attributable to endothelium-derived hyperpolarisation (EDH). In conclusion, sympatho-adrenal overactivation caused by high salt intake and insulin resistance evoked coronary microvessel endothelial dysfunction and diminished NO sensitivity, which were restored by MET and CAR treatment in spite of ongoing inflammation and oxidative-nitrosative stress presumably caused by uninhibited renin-angiotensin-aldosterone system (RAAS) overactivation.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Carvedilol/farmacologia , Endotélio Vascular/efeitos dos fármacos , Resistência à Insulina , Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Animais , Angiografia Coronária , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Hipertensão/fisiopatologia , Masculino , Metoprolol/farmacologia , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Cloreto de Sódio na Dieta/administração & dosagemRESUMO
Recently, veno-venous extracorporeal membrane oxygenation (V-V ECMO) has been commonly used in the world to support patients with severe respiratory failure. However, V-V ECMO is a new technology compared to veno-arterial extracorporeal membrane oxygenation and cardiopulmonary bypass, and there are few reports of basic research. Although continuing research is desired, clinical research that standardizes conditions such as patients' background characteristics is difficult. The purpose of this study was to establish a simple and stably maintainable miniature V-V ECMO model to study the mechanisms of the biological reactions in circulation during V-V ECMO. The V-V ECMO system consisted of an original miniature membrane oxygenator, polyvinyl chloride tubing line, and roller pump. The priming volume of this system was only 8 mL. Polyethylene tubing was used to cannulate the right femoral vein as the venous return cannula for the V-V ECMO system. A 16-G cannula was passed through the right internal jugular vein and advanced into the right atrium as the conduit for venous uptake. The animals were divided into 2 groups: SHAM group and V-V ECMO group. V-V ECMO was initiated and maintained at 50-60 mL/kg/min, and oxygen was added into the oxygenator during V-V ECMO at a concentration of 100% (pump flow:oxygen = 1:10). Blood pressure was measured continuously, and blood cells were measured by blood collection. During V-V ECMO, the blood pressure and hemodilution rate were maintained around 80 mm Hg and 20%, respectively. Hb was kept at >10 g/dL, and V-V ECMO could be maintained without blood transfusion. It was possible to confirm oxygenation of and carbon dioxide removal from the blood. Likewise, the pH was adequately maintained. There were no problems with this miniature V-V ECMO system, and extracorporeal circulation progressed safely. In this study, a novel miniature V-V ECMO model was established in the rat. A miniature V-V ECMO model appears to be very useful for studying the mechanisms of the biological reactions during V-V ECMO and to perform basic studies of circulation assist devices.
Assuntos
Oxigenação por Membrana Extracorpórea/métodos , Modelos Animais , Oxigenadores de Membrana , Animais , Cânula , Desenho de Equipamento , Oxigenação por Membrana Extracorpórea/instrumentação , Masculino , RatosRESUMO
The management of heart failure patients presenting in a moribund state remains challenging, despite significant advances in the field of ventricular assist systems. Bridge to decision involves using temporary devices to stabilize the hemodynamic state of such patients while further assessment is performed and a decision can be made regarding patient management. We developed a new temporary left ventricular assist system employing a disposable centrifugal pump with a hydrodynamically levitated bearing. We used three adult goats (body weight, 58-68 kg) to investigate the 30-day performance and hemocompatibility of the newly developed left ventricular assist system, which included the pump, inflow and outflow cannulas, the extracorporeal circuit, and connectors. Hemodynamic, hematologic, and blood chemistry measurements were investigated as well as end-organ effect on necropsy. All goats survived for 30 days in good general condition. The blood pump was operated at a rotational speed of 3000-4500 rpm and a mean pump flow of 3.2 ± 0.6 L min. Excess hemolysis, observed in one goat, was due to the inadequate increase in pump rotational speed in response to drainage insufficiency caused by continuous contact of the inflow cannula tip with the left ventricular septal wall in the early days after surgery. At necropsy, no thrombus was noted in the pump, and no damage caused by mechanical contact was found on the bearing. The newly developed temporary left ventricular assist system using a disposable centrifugal pump with hydrodynamic bearing demonstrated consistent and satisfactory hemodynamic performance and hemocompatibility in the goat model.
Assuntos
Insuficiência Cardíaca/cirurgia , Ventrículos do Coração/cirurgia , Coração Auxiliar , Hemodinâmica/fisiologia , Animais , Modelos Animais de Doenças , Cabras , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/fisiopatologia , Desenho de PróteseRESUMO
Chronic intermittent hypoxia (IH) induces oxidative stress and inflammation, which impair vascular endothelial function. Long-term insulin resistance also leads to endothelial dysfunction. We determined, in vivo, whether the effects of chronic IH and insulin resistance on endothelial function augment each other. Male 12-wk-old Goto-Kakizaki (GK) and Wistar control rats were subjected to normoxia or chronic IH (90-s N2, 5% O2 at nadir, 90-s air, 20 cycles/h, 8 h/day) for 4 wk. Coronary endothelial function was assessed using microangiography with synchrotron radiation. Imaging was performed at baseline, during infusion of acetylcholine (ACh, 5 µg·kg(-1)·min(-1)) and then sodium nitroprusside (SNP, 5 µg·kg(-1)·min(-1)), after blockade of both nitric oxide (NO) synthase (NOS) with N(ω)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg) and cyclooxygenase (COX, meclofenamate, 3 mg/kg), and during subsequent ACh. In GK rats, coronary vasodilatation in response to ACh and SNP was blunted compared with Wistar rats, and responses to ACh were abolished after blockade. In Wistar rats, IH blunted the ability of ACh or SNP to increase the number of visible vessels. In GK rats exposed to IH, neither ACh nor SNP were able to increase visible vessel number or caliber, and blockade resulted in marked vasoconstriction. Our findings indicate that IH augments the deleterious effects of insulin resistance on coronary endothelial function. They appear to increase the dependence of the coronary microcirculation on NO and/or vasodilator prostanoids, and greatly blunt the residual vasodilation in response to ACh after blockade of NOS/COX, presumably mediated by endothelium-derived hyperpolarizing factors.
Assuntos
Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/fisiopatologia , Hipóxia/complicações , Hipóxia/fisiopatologia , Resistência à Insulina , Microcirculação , Animais , Doença Crônica , Doença da Artéria Coronariana/diagnóstico por imagem , Circulação Coronária , Progressão da Doença , Hipóxia/diagnóstico por imagem , Masculino , Ratos , Ratos WistarRESUMO
Cell-sheet transplantation induces angiogenesis for chronic myocardial infarction (MI), though insufï¬cient capillary maturation and paucity of arteriogenesis may limit its therapeutic effects. Omentum has been used clinically to promote revascularization and healing of ischemic tissues. We hypothesized that cell-sheet transplantation covered with an omentum-flap would effectively establish mature blood vessels and improve coronary microcirculation physiology, enhancing the therapeutic effects of cell-sheet therapy. Rats were divided into four groups after coronary ligation; skeletal myoblast cell-sheet plus omentum-flap (combined), cell-sheet only, omentum-flap only, and sham operation. At 4 weeks after the treatment, the combined group showed attenuated cardiac hypertrophy and fibrosis, and a greater amount of functionally (CD31(+)/lectin(+)) and structurally (CD31(+)/α-SMA(+)) mature blood vessels, along with myocardial upregulation of relevant genes. Synchrotron-based microangiography revealed that the combined procedure increased vascularization in resistance arterial vessels with better dilatory responses to endothelium-dependent agents. Serial (13)N-ammonia PET showed better global coronary flow reserve in the combined group, mainly attributed to improvement in the basal left ventricle. Consequently, the combined group had sustained improvements in cardiac function parameters and better functional capacity. Cell-sheet transplantation with an omentum-flap better promoted arteriogenesis and improved coronary microcirculation physiology in ischemic myocardium, leading to potent functional recovery in the failing heart.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Circulação Coronária , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/terapia , Neovascularização Fisiológica , Omento , Animais , Movimento Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Expressão Gênica , Sobrevivência de Enxerto , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Hemodinâmica , Infarto do Miocárdio/complicações , Miocárdio/metabolismo , Miocárdio/patologia , Ratos , Fluxo Sanguíneo Regional , Transplantes , Remodelação Vascular , Função Ventricular EsquerdaRESUMO
We previously developed a novel control system for a continuous-flow left ventricular assist device (LVAD), the EVAHEART, and demonstrated that sufficient pulsatility can be created by increasing its rotational speed in the systolic phase (pulsatile mode) in a normal heart animal model. In the present study, we assessed this system in its reliability and ability to follow heart rate variability. We implanted an EVAHEART via left thoracotomy into five goats for the Study for Fixed Heart Rate with ventricular pacing at 80, 100, 120 and 140 beats/min and six goats for the Study for native heart rhythm. We tested three modes: the circuit clamp, the continuous mode and the pulsatile mode. In the pulsatile mode, rotational speed was increased during the initial 35 % of the RR interval by automatic control based on the electrocardiogram. Pulsatility was evaluated by pulse pressure and dP/dt max of aortic pressure. As a result, comparing the pulsatile mode with the continuous mode, the pulse pressure was 28.5 ± 5.7 vs. 20.3 ± 7.9 mmHg, mean dP/dt max was 775.0 ± 230.5 vs 442.4 ± 184.7 mmHg/s at 80 bpm in the study for fixed heart rate, respectively (P < 0.05). The system successfully determined the heart rate to be 94.6 % in native heart rhythm. Furthermore, pulse pressure was 41.5 ± 7.9 vs. 27.8 ± 5.6 mmHg, mean dP/dt max was 716.2 ± 133.9 vs 405.2 ± 86.0 mmHg/s, respectively (P < 0.01). In conclusion, our newly developed the pulsatile mode for continuous-flow LVADs reliably provided physiological pulsatility with following heart rate variability.
Assuntos
Insuficiência Cardíaca/terapia , Frequência Cardíaca , Coração Auxiliar , Fluxo Pulsátil , Animais , Pressão Sanguínea , Modelos Animais de Doenças , Eletrocardiografia , Cabras , Coração/fisiologia , Reprodutibilidade dos Testes , SístoleRESUMO
For the continued development of improved mechanical circulatory systems, longer term evaluation of new devices in animal model experiments may be critical. The effects of anticoagulants in adult goats have not been well studied. We assessed the effects of oral warfarin in three adult goats during fasting or after feeding. The goats [weighing 57.8 ± 8.1 kg (53.0-67.2 kg)] were administered warfarin orally beginning at a dose of 5 mg/day and then increasing to 10, 20, 40, and 60 mg every 2 weeks. One goat (receiving 10 mg/day warfarin) was killed on day 27 because of the inability to stand. After administration of 60 mg warfarin, the remaining goat received no warfarin for 4 days to return to coagulated state. The goats were then fasted and treated with 40 mg warfarin. During warfarin administration, both goats required a dose of 60 mg/day to achieve International Normalized Ratios (INRs) of approximately 2.5; however, when, the animals were in the fasted condition, precipitous extension of INR was observed in 5 days. After resuming feeding, the INR was reduced to the proper range. We showed the tendency that warfarin therapy in goats required higher doses than the doses administered to human patients and that the effects of therapy were related to the feeding state. The results of this study provide important information for development of anticoagulation protocols to assess mechanical circulatory support devices for long-term use in preclinical examination.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Jejum/sangue , Varfarina/farmacologia , Administração Oral , Animais , Esquema de Medicação , Feminino , Cabras , Coeficiente Internacional Normatizado , Masculino , Estado NutricionalRESUMO
BACKGROUND: Impaired actin-myosin cross-bridge (CB) dynamics correlate with impaired left ventricular (LV) function in early diabetic cardiomyopathy (DCM). Elevated expression and activity of Rho kinase (ROCK) contributes to the development of DCM. ROCK targets several sarcomeric proteins including myosin light chain 2, myosin binding protein-C (MyBP-C), troponin I (TnI) and troponin T that all have important roles in regulating CB dynamics and contractility of the myocardium. Our aim was to examine if chronic ROCK inhibition prevents impaired CB dynamics and LV dysfunction in a rat model of early diabetes, and whether these changes are associated with changes in myofilament phosphorylation state. METHODS: Seven days post-diabetes induction (65 mg/kg ip, streptozotocin), diabetic rats received the ROCK inhibitor, fasudil (10 mg/kg/day ip) or vehicle for 14 days. Rats underwent cardiac catheterization to assess LV function simultaneous with X-ray diffraction using synchrotron radiation to assess in situ CB dynamics. RESULTS: Compared to controls, diabetic rats developed mild systolic and diastolic dysfunction, which was attenuated by fasudil. End-diastolic and systolic myosin proximity to actin filaments were significantly reduced in diabetic rats (P < 0.05). In all rats there was an inverse correlation between ROCK1 expression and the extension of myosin CB in diastole, with the lowest ROCK expression in control and fasudil-treated diabetic rats. In diabetic and fasudil-treated diabetic rats changes in relative phosphorylation of TnI and MyBP-C were not significant from controls. CONCLUSIONS: Our results demonstrate a clear role for ROCK in the development of LV dysfunction and impaired CB dynamics in early DCM.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Cardiomiopatias Diabéticas/prevenção & controle , Contração Miocárdica/efeitos dos fármacos , Miocárdio/enzimologia , Miosinas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Disfunção Ventricular Esquerda/prevenção & controle , Função Ventricular Esquerda/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Actinas/metabolismo , Animais , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Cardiomiopatias Diabéticas/enzimologia , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/fisiopatologia , Masculino , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Disfunção Ventricular Esquerda/enzimologia , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/fisiopatologia , Quinases Associadas a rho/metabolismoRESUMO
Extracorporeal circulation (ECC) is indispensable for cardiac surgery. Despite the fact that ECCcauses damage to blood components and is non-physiologic, its pathophysiology has not been fully elucidated. This is because difficulty in clinical research and animal experiments keeps the knowledge insufficient. Therefore, it is desirable to have a miniature ECC model for small animals, which enables repetitive experiments, to study the mechanism of pathophysiological changes during ECC. We developed a miniature ECC system and applied it to the rat. We measured changes in hemodynamics, blood gases and hemoglobin (Hb) concentration, serum cytokines (TNF-α, IL-6, IL-10), biochemical markers (LDH, AST, ALT), and the wet-to-dry weight (W/D) ratio of the lung for assessing whether the rat ECC model is comparable to the human ECC. The ECC system consisted of a membranous oxygenator (polypropylene, 0.03 m(2)), tubing line (polyvinyl chloride), and roller pump. Priming volume of this system is only 8 ml. Rats (400-450 g) were divided into the SHAM group (n = 7) and the ECC group (n = 7). Blood samples were collected before, 60 and 120 min after initiation of ECC. During ECC, blood pressure and Hb were maintained around 80 mmHg and 10 g/dL, respectively. The levels of the inflammatory and biochemical markers and the W/D ratio were significantly elevated in the ECC group, indicating some organ damages and systemic inflammatory responses during ECC. We successfully established the ECC for the rat. This miniature ECC model could be a useful approach for studying the mechanism of pathophysiology during ECC and basic assessment of the ECC devices.
Assuntos
Ponte Cardiopulmonar/métodos , Circulação Extracorpórea/métodos , Animais , Biomarcadores/sangue , Interleucina-10/sangue , Interleucina-6/sangue , Masculino , Modelos Animais , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/sangueRESUMO
The clinical use of veno-venous extracorporeal membrane oxygenation (VVECMO) in adult patients with respiratory failure is rapidly increasing. However, recirculation of blood oxygenated by ECMO back into the circuit may occur in VVECMO, resulting in insufficient oxygenation. The cannula position and bypass flow rate are two major factors influencing recirculation, but the relationship and ideal configuration of these factors are not fully understood. In the present study, we attempted to clarify these parameters for effective gas exchange. VVECMO was performed in eight adult goats under general anesthesia. The position of the drainage cannula was fixed in the inferior vena cava (IVC), but the return cannula position was varied between the IVC, right atrium (RA), and superior vena cava (SVC). At each position, the recirculation rates calculated, and the adequacy of oxygen delivery by ECMO in supplying systemic oxygen demand was assessed by measuring the arterial oxygen saturation (SaO2) and pressure (PaO2). Although the recirculation rates increased as the bypass flow rates increased, SaO2 and PaO2 also increased in any position of return cannula. The recirculation rates and PaO2 were 27 ± 2% and 162 ± 16 mmHg, 36 ± 6% and 139 ± 11 mmHg, and 63 ± 6% and 77 ± 9 mmHg in the SVC, RA and IVC position at 4 L/min respectively. In conclusion, the best return cannula position was the SVC, and a high bypass flow rate was advantageous for effective oxygenation. Both the bypass flow rates and cannula position must be considered to achieve effective oxygenation.
Assuntos
Cateterismo Cardíaco , Oxigenação por Membrana Extracorpórea , Troca Gasosa Pulmonar/fisiologia , Insuficiência Respiratória/terapia , Adulto , Animais , Catéteres , Modelos Animais de Doenças , Drenagem , Oxigenação por Membrana Extracorpórea/instrumentação , Feminino , Cabras , Humanos , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica , OxigênioRESUMO
Chronic intermittent hypoxia (IH) provokes a centrally mediated increase in sympathetic nerve activity (SNA). Although this sympathetic hyperexcitation has been linked to systemic hypertension, its effect on the pulmonary vasculature is unclear. This study aimed to assess IH-mediated sympathetic excitation in modulating pulmonary vasculature tone, particularly acute hypoxia vasoconstrictor response (HPV), and the central ß-adrenergic signaling pathway for facilitating the increase in SNA. Sprague-Dawley rats were exposed to IH (cycle of 4% O2 for 90 s/air for 90 s) for 8 h/day for 6 weeks. Subsequently, rats were anesthetized and either pulmonary SNA was recorded (electrophysiology), or the pulmonary vasculature was visualized using microangiography. Pulmonary sympathetic and vascular responses to acute hypoxia were assessed before and after central ß1-adrenergic receptor blockade (Metoprolol, 200 nmol i.c.v.). Chronic IH increased baseline SNA (110% increase), and exacerbated the sympathetic response to acute hypoxia. Moreover, the magnitude of HPV in IH rats was blunted compared to control rats (e.g., 10 and 20% vasoconstriction, respectively). In only the IH rats, ß1-receptor blockade with metoprolol attenuated the hypoxia-induced increase in pSNA and exacerbated the magnitude of acute HPV, so that both sympathetic and HPV responses were similar to that of control rats. Interestingly, the expression of ß1-receptors within the brainstem was similar between both control and IH rats. These results suggest that the centrally mediated increase in SNA following IH acts to blunt the local vasoconstrictor effect of acute hypoxia, which reflects an inherent difference between vasodilator and vasoconstrictor actions of SNA in pulmonary and systemic circulations.
Assuntos
Hipóxia/fisiopatologia , Pulmão/fisiopatologia , Fenômenos Fisiológicos Respiratórios , Sistema Nervoso Simpático/fisiopatologia , Angiografia/métodos , Animais , Western Blotting , Eletrofisiologia , Pulmão/irrigação sanguínea , Pulmão/inervação , Masculino , Ratos , Ratos Sprague-Dawley , Vasoconstrição/fisiologiaRESUMO
BACKGROUND: Arsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death. METHODS: HL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression. RESULTS: HL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO. CONCLUSIONS: The enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Arsenicais/administração & dosagem , Butionina Sulfoximina/administração & dosagem , Leucemia Mieloide Aguda/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo/fisiologia , Óxidos/administração & dosagem , Proteínas Proto-Oncogênicas/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Trióxido de Arsênio , Proteína 11 Semelhante a Bcl-2 , Combinação de Medicamentos , Técnicas de Silenciamento de Genes/métodos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/patologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genéticaRESUMO
Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150 kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a ß-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N'-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.
Assuntos
Domínio Catalítico , Quitinases/genética , Quitinases/metabolismo , Paenibacillus/enzimologia , Sequência de Aminoácidos , Quitina/metabolismo , Quitinases/química , Quitinases/isolamento & purificação , Escherichia coli/genética , Expressão Gênica , Dados de Sequência Molecular , Mutagênese , Mutação , Especificidade por SubstratoRESUMO
Right ventricular (RV) failure is a potentially fatal complication after treatment with a left ventricular assist device (LVAD). Ventricular septal shift caused by such devices is an important factor in the progress of RV dysfunction. We developed a control system for a rotary blood pump that can change rotational speed (RS) in synchrony with the cardiac cycle. We postulated that decreasing systolic RS using this system would alter ventricular septal movement and thus prevent RV failure. We implanted the EVAHEART ventricular assist device into seven adult goats weighing 54.1 ± 2.1 kg and induced acute bi-ventricular dysfunction by coronary embolization. Left and RV pressure was monitored, and ventricular septal movement was echocardiographically determined. We evaluated circuit-clamp mode as the control condition, as well as continuous and counter-pulse modes, both with full bypass. As a result, a leftward ventricular septal shift occurred in continuous and counter-pulse modes. The septal shift was corrected as a result of decreased RS during the systolic phase in counter-pulse mode. RV fractional area change improved in counter-pulse (59.0 ± 4.6%) compared with continuous (44.7 ± 4.0%) mode. In conclusion, decreased RS delivered during the systolic phase using the counter-pulse mode of our new system holds promise for the clinical correction of ventricular septal shift resulting from a LVAD and might confer a benefit upon RV function.
Assuntos
Contrapulsação/instrumentação , Coração Auxiliar/efeitos adversos , Disfunção Ventricular Direita/etiologia , Disfunção Ventricular Direita/prevenção & controle , Animais , Modelos Animais de Doenças , Cabras , Rotação , Sístole/fisiologia , Ultrassonografia , Disfunção Ventricular Direita/diagnóstico por imagem , Septo Interventricular/diagnóstico por imagem , Septo Interventricular/fisiopatologiaRESUMO
In recent years, venovenous extracorporeal membrane oxygenation (VV ECMO) has been used to support patients with severe lung disease. Active use of VV ECMO was also recommended for severe respiratory failure due to COVID-19. However, VV ECMO is also known to cause various complications due to extracorporeal circulation. Although we conducted ECMO research using rats, we have not been able to establish whether double-lumen single-cannulation VV ECMO models in rats have been described previously. The purpose of this study was to establish a simple, stable, and maintainable miniature double-lumen single-canulation VV ECMO model in rats. A double-lumen catheter used as a plain central venous catheter (SMAC plus Seldinger type; Covidien Japan Co., Tokyo, Japan) was passed through the right external jugular vein and advanced into the right atrium as a conduit for venous uptake. The VV ECMO system comprised a roller pump, miniature membrane oxygenator, and polyvinyl chloride tubing line. During VV ECMO, blood pressure and hemodilution rate were maintained at around 80 mmHg and 30%, respectively. Hemoglobin was kept at >9 g/dL, no serious hemolysis was observed, and VV ECMO was maintained without blood transfusion. Oxygenation and removal of carbon dioxide from the blood were confirmed and pH was adequately maintained. This miniature VV ECMO model appears very useful for studying the mechanisms of biological reactions during VV ECMO.
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Introduction Prolonged sitting-induced blood pooling in the lower legs can increase blood pressure through increased sympathetic nerve activity and peripheral vascular resistance, an aspect that has been understudied as a primary outcome. This study compared the effects of prolonged sitting with those of prolonged supination on blood pressure in healthy young men. Methods This randomized crossover study included 16 healthy young men (mean age: 21.6 ± 0.7 years) who were randomly assigned to a three-hour supine (CON) or three-hour sitting (SIT) condition, followed by a washout period of at least one week. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), heart rate (HR), low-frequency/high-frequency (LF/HF) ratio derived from heart rate variability, and leg circumference were measured at 60, 120, and 180 minutes from baseline. These indices were compared by two-way (time × conditions) analysis of variance (ANOVA). Results In the SIT condition, DBP, MAP, HR, LF/HF ratio, and leg circumference increased significantly over time (P < 0.05) and were significantly higher than those in the CON condition (P < 0.05). However, SBP showed no significant change over time and between conditions. Conclusions The findings indicate the involvement of sympathetic nerve activity and increased peripheral vascular resistance induced by fluid retention in the lower legs with increased DBP and MAP in healthy young men.
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Diabetes is independently associated with a specific cardiomyopathy, characterized by impaired cardiac muscle relaxation and force development. Using synchrotron radiation small-angle x-ray scattering, this study investigated in the in situ heart and in real-time whether changes in cross-bridge disposition and myosin interfilament spacing underlie the early development of diabetic cardiomyopathy. Experiments were conducted using anesthetized Sprague-Dawley rats 3 weeks after treatment with either vehicle (control) or streptozotocin (diabetic). Diffraction patterns were recorded during baseline and dobutamine infusions simultaneous with ventricular pressure-volumetry. From these diffraction patterns myosin mass transfer to actin filaments was assessed as the change in intensity ratio (I(1,0)/I(1,1)). In diabetic hearts cross-bridge disposition was most notably abnormal in the diastolic phase (p < 0.05) and to a lesser extent the systolic phase (p < 0.05). In diabetic rats only, there was a transmural gradient of contractile depression. Elevated diabetic end-diastolic intensity ratios were correlated with the suppression of diastolic function (p < 0.05). Furthermore, the expected increase in myosin head transfer by dobutamine was significantly blunted in diabetic animals (p < 0.05). Interfilament spacing did not differ between groups. We reveal that impaired cross-bridge disposition and radial transfer may thus underlie the early decline in ventricular function observed in diabetic cardiomyopathy.