RESUMEN
Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4-/- mouse model of Leigh syndrome, continuously breathing 11% O2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4-/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4-/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4-/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O2. Compared to WT control mice, Ndufs4-/- mice breathing air have reduced brain O2 consumption as evidenced by an elevated partial pressure of O2 in IJV blood (PijvO2) despite a normal PO2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4-/- mice, hypoxia treatment normalized the cerebral venous PijvO2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD+) were lower in Ndufs4-/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O2) has been shown to be an ineffective therapy for Ndufs4-/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD+ deficiency may hold promise for treating Leigh syndrome.
Asunto(s)
Encéfalo/metabolismo , Complejo I de Transporte de Electrón/genética , Enfermedad de Leigh/metabolismo , NAD/genética , Oxígeno/metabolismo , Animales , Encéfalo/patología , Hipoxia de la Célula/fisiología , Modelos Animales de Enfermedad , Complejo I de Transporte de Electrón/metabolismo , Humanos , Enfermedad de Leigh/genética , Enfermedad de Leigh/terapia , Metabolómica , Ratones , Mitocondrias , NAD/deficiencia , Enfermedades Neurodegenerativas , Respiración/genéticaRESUMEN
Hypoxic pulmonary vasoconstriction (HPV) is a physiological vasomotor response that maintains systemic oxygenation by matching perfusion to ventilation during alveolar hypoxia. Although mitochondria appear to play an essential role in HPV, the impact of mitochondrial dysfunction on HPV remains incompletely defined. Mice lacking the mitochondrial complex I (CI) subunit Ndufs4 ( Ndufs4-/-) develop a fatal progressive encephalopathy and serve as a model for Leigh syndrome, the most common mitochondrial disease in children. Breathing normobaric 11% O2 prevents neurological disease and improves survival in Ndufs4-/- mice. In this study, we found that either genetic Ndufs4 deficiency or pharmacological inhibition of CI using piericidin A impaired the ability of left mainstem bronchus occlusion (LMBO) to induce HPV. In mice breathing air, the partial pressure of arterial oxygen during LMBO was lower in Ndufs4-/- and in piericidin A-treated Ndufs4+/+ mice than in respective controls. Impairment of HPV in Ndufs4-/- mice was not a result of nonspecific dysfunction of the pulmonary vascular contractile apparatus or pulmonary inflammation. In Ndufs4-deficient mice, 3 wk of breathing 11% O2 restored HPV in response to LMBO. When compared with Ndufs4-/- mice breathing air, chronic hypoxia improved systemic oxygenation during LMBO. The results of this study show that, when breathing air, mice with a congenital Ndufs4 deficiency or chemically inhibited CI function have impaired HPV. Our study raises the possibility that patients with inborn errors of mitochondrial function may also have defects in HPV.
Asunto(s)
Complejo I de Transporte de Electrón/deficiencia , Hipoxia/fisiopatología , Enfermedad de Leigh/fisiopatología , Vasoconstricción/fisiología , Animales , Bronquios/metabolismo , Modelos Animales de Enfermedad , Hipoxia/metabolismo , Pulmón/metabolismo , Pulmón/fisiopatología , Ratones Transgénicos , Mitocondrias/metabolismo , Arteria Pulmonar/metabolismo , Circulación Pulmonar/fisiologíaRESUMEN
The nitric oxide - guanylyl cyclase-1 - cyclic guanylate monophosphate (NO-GC-1-cGMP) pathway has emerged as a potential pathogenic mechanism for glaucoma, a common intraocular pressure (IOP)-related optic neuropathy characterized by the degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. NO activates GC-1 to increase cGMP levels, which are lowered by cGMP-specific phosphodiesterase (PDE) activity. This pathway appears to play a role in both the regulation of IOP, where reduced cGMP levels in mice leads to elevated IOP and subsequent RGC degeneration. Here, we investigated whether potentiation of cGMP signaling could protect RGCs from glaucomatous degeneration. We administered the PDE5 inhibitor tadalafil orally (10â¯mg/kg/day) in murine models of two forms of glaucoma - primary open angle glaucoma (POAG; GC-1-/- mice) and primary angle-closure glaucoma (PACG; Microbead Occlusion Model) - and measured RGC viability at both the soma and axon level. To determine the direct effect of increased cGMP on RGCs in vitro, we treated axotomized whole retina and primary RGC cultures with the cGMP analogue 8-Br-cGMP. Tadalafil treatment increased plasma cGMP levels in both models, but did not alter IOP or mean arterial pressure. Nonetheless, tadalafil treatment prevented degeneration of RGC soma and axons in both disease models. Treatment of whole, axotomized retina and primary RGC cultures with 8-Br-cGMP markedly attenuated both necrotic and apoptotic cell death pathways in RGCs. Our findings suggest that enhancement of the NO-GC-1-cGMP pathway protects the RGC body and axon in murine models of POAG and PACG, and that enhanced signaling through this pathway may serve as a novel glaucoma treatment, acting independently of IOP.
Asunto(s)
GMP Cíclico/metabolismo , Glaucoma/metabolismo , Degeneración Retiniana/metabolismo , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Glaucoma/prevención & control , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Ratones Noqueados , Inhibidores de Fosfodiesterasa 5/administración & dosificación , Ratas Sprague-Dawley , Degeneración Retiniana/prevención & control , Células Ganglionares de la Retina/efectos de los fármacos , Transducción de Señal , Tadalafilo/administración & dosificaciónRESUMEN
BACKGROUND: Hypoxic pulmonary vasoconstriction (HPV) optimizes the match between ventilation and perfusion in the lung by reducing blood flow to poorly ventilated regions. Sepsis and endotoxemia impair HPV. We previously showed that nitric oxide synthase 2 (NOS2) is required, but not sufficient, for the effect of endotoxin on HPV. The aim of the current study was to identify additional factors that might contribute to the impairment of HPV during endotoxemia. METHODS: Gene expression profiling was determined using pulmonary tissues from NOS2-deficient (NOS2-/-) and wild-type mice subjected to endotoxin or saline challenge (control). HPV was accessed as the percentage increase in left pulmonary vascular resistance (LPVR) in response to left main bronchus occlusion (LMBO) in wild-type mice. RESULTS: Among the 22,690 genes analyzed, endotoxin induced a greater than three-fold increase in 59 and 154 genes in the lungs of wild-type and NOS2-/- mice, respectively. Of all the genes induced by endotoxin in wild-type mice, arginase 1 (Arg1) showed the greatest increase (16.3-fold compared to saline treated wild-type mice). In contrast, endotoxin did not increase expression of Arg1 in NOS2-/- mice. There was no difference in the endotoxin-induced expression of Arg2 between wild-type and NOS2-deficient mice. We investigated the role of arginase in HPV by treating the mice with normal saline or the arginase inhibitor Nω-hydroxy-nor-L-arginine (norNOHA). In control mice (in the absence of endotoxin) treated with normal saline, HPV was intact as determined by profound LMBO-induced increase in LPVR (121 ± 22% from baseline). During endotoxemia and treatment with normal saline, HPV was impaired compared to normal saline treated control mice (33 ± 9% vs. 121 ± 22%, P < 0.05). HPV was restored in endotoxin-exposed mice after treatment with the arginase inhibitor norNOHA as shown by the comparison to endotoxemic mice treated with normal saline (113 ± 29% vs, 33 ± 9%, P < 0.05) and to control mice treated with normal saline (113 ± 29% vs, 121 ± 22%, P = 0.97). CONCLUSIONS: The results of this study suggest that endotoxemia induces Arg1 and that arginase contributes to the endotoxin-induced impairment of HPV in mice.
Asunto(s)
Arginasa/metabolismo , Endotoxemia/enzimología , Circulación Pulmonar/fisiología , Resistencia Vascular/fisiología , Vasoconstricción/fisiología , Animales , Endotoxemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Sickle cell disease is an inherited disorder of hemoglobin (Hb). During a sickle cell crisis, deoxygenated sickle hemoglobin (deoxyHbS) polymerizes to form fibers in red blood cells (RBCs), causing the cells to adopt "sickled" shapes. Using small molecules to increase the affinity of Hb for oxygen is a potential approach to treating sickle cell disease, because oxygenated Hb interferes with the polymerization of deoxyHbS. We have identified a triazole disulfide compound (4,4'-di(1,2,3-triazolyl)disulfide, designated TD-3), which increases the affinity of Hb for oxygen. The crystal structures of carboxy- and deoxy-forms of human adult Hb (HbA), each complexed with TD-3, revealed that one molecule of the monomeric thiol form of TD-3 (5-mercapto-1H-1,2,3-triazole, designated MT-3) forms a disulfide bond with ß-Cys93, which inhibits the salt-bridge formation between ß-Asp94 and ß-His146. This inhibition of salt bridge formation stabilizes the R-state and destabilizes the T-state of Hb, resulting in reduced magnitude of the Bohr effect and increased affinity of Hb for oxygen. Intravenous administration of TD-3 (100 mg/kg) to C57BL/6 mice increased the affinity of murine Hb for oxygen, and the mice did not appear to be adversely affected by the drug. TD-3 reduced in vitro hypoxia-induced sickling of human sickle RBCs. The percentage of sickled RBCs and the P50 of human SS RBCs by TD-3 were inversely correlated with the fraction of Hb modified by TD-3. Our study shows that TD-3, and possibly other triazole disulfide compounds that bind to Hb ß-Cys93, may provide new treatment options for patients with sickle cell disease.
Asunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Antidrepanocíticos/farmacología , Disulfuros/farmacología , Eritrocitos/efectos de los fármacos , Hemoglobinas/metabolismo , Oxígeno/metabolismo , Triazoles/farmacología , Anemia de Células Falciformes/metabolismo , Animales , Eritrocitos/metabolismo , Hemoglobina Falciforme/metabolismo , Humanos , Hipoxia/tratamiento farmacológico , Hipoxia/metabolismo , Metalotioneína 3 , Ratones , Ratones Endogámicos C57BL , Polimerizacion/efectos de los fármacos , Unión ProteicaRESUMEN
OBJECTIVES: To test the safety of a novel miniaturized device that produces nitric oxide (NO) from air by pulsed electrical discharge, and to demonstrate that the generated NO can be used to vasodilate the pulmonary vasculature in rabbits with chemically-induced pulmonary hypertension. STUDY DESIGN: A miniature NO (mini-NO) generator was tested for its ability to produce therapeutic levels (20-80â¯parts per million (ppm)) of NO, while removing potentially toxic gases and metal particles. We studied healthy 6-month-old New Zealand rabbits weighing 3.4⯱â¯0.4â¯kg (mean⯱â¯SD, nâ¯=â¯8). Pulmonary hypertension was induced by chemically increasing right ventricular systolic pressure to 28-30â¯mmHg. The mini-NO generator was placed near the endotracheal tube. Production of NO was triggered by a pediatric airway flowmeter during the first 0.5â¯s of inspiration. RESULTS: In rabbits with acute pulmonary hypertension, the mini-NO generator produced sufficient NO to induce pulmonary vasodilation. Potentially toxic nitrogen dioxide (NO2) and ozone (O3) were removed by the Ca(OH)2 scavenger. Metallic particles, released from the electrodes by the electric plasma, were removed by a 0.22⯵m filter. While producing 40â¯ppm NO, the mini-NO generator was cooled by a flow of air (70â¯ml/min) and the external temperature of the housing did not exceed 31⯰C. CONCLUSIONS: The mini-NO generator safely produced therapeutic levels of NO from air. The mini-NO generator is an effective and economical approach to producing NO for treating neonatal pulmonary hypertension and will increase the accessibility and therapeutic uses of life-saving NO therapy worldwide.
Asunto(s)
Hidróxido de Calcio/administración & dosificación , Hipertensión Pulmonar/tratamiento farmacológico , Óxido Nítrico/administración & dosificación , Terapia Respiratoria/instrumentación , Administración por Inhalación , Animales , Diseño de Equipo , Femenino , Masculino , Metales/aislamiento & purificación , Óxido Nítrico/uso terapéutico , Conejos , Temperatura , Presión Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: Digitalization in the health system is a topic that is rapidly gaining popularity, and not only because of the current pandemic. As in many areas of daily life, digitalization is becoming increasingly important in the medical field amid the exponential rise in the use of computers and smartphones. This opens up new possibilities for optimizing patient education in the context of anesthesia. The main aim of this study was to assess the implementation of remote consent in Europe. METHODS: An online survey entitled "Digital online Patient Informed Consent for Anesthesia before Elective Surgery. Recent practice in Europe," with a total of 27 questions, was sent by the European Society of Anesthesiology and Intensive Care (ESAIC) to their members in 47 European countries. To assess the effect of the economy on digitalization and legal status with regard to anesthesia consent, data were stratified based on gross domestic product per capita (GDPPC). RESULTS: In total, 23.1% and 37.2% of the 930 participants indicated that it was possible to obtain consent online or via telephone, respectively. This observation was more often reported in countries with high GDPPC levels than in countries with low GDPPC levels. Furthermore, 27.3% of the responses for simple anesthesia, 18.7% of the responses for complex anesthesia, and 32.2% of the responses for repeated anesthesia indicated that remote consent was in accordance with the law, and this was especially prevalent in countries with high GDPPC. Concerning the timing of consent, patients were informed at least one day before in 67.1% of cases for simple procedures and in 85.2% of cases for complex procedures. CONCLUSION: Even European countries with high GDPPC use remote informed consent only in a minority of cases, and most of the time for repeated anesthetic procedures. This might reflect the inconsistent legal situation and inhomogeneous medical technical structures across Europe.
RESUMEN
BACKGROUND: One undisputed benefit of digital support is the possibility of contact reduction, which has become particularly important in the context of the COVID-19 pandemic. However, to the best of our knowledge, there is currently no study assessing the Europe-wide use of digital online pre-operative patient information or evaluation in the health sector. The aim of this study was to give an overview of the current status in Europe. METHODS: A web-based questionnaire covering the informed consent process was sent to members of the European Society of Anaesthesia and Intensive Care Medicine (ESAIC) in 47 European countries (42,433 recipients/930 responses). Six questions related specifically to the practice in paediatrics. RESULTS: A total of 70.2% of the respondents indicated that it was not possible to obtain informed consent via the Internet in a routine setting, and 67.3% expressed that they did not know whether it is in line with the legal regulations. In paediatric anaesthesia, the informed consent of only one parent was reported to be sufficient by 77.6% of the respondents for simple interventions and by 63.8% for complex interventions. Just over 50% of the respondents judged that proof of identity of the parents was necessary, but only 29.9% stated that they ask for it in clinical routine. In the current situation, 77.9% would favour informed consent in person, whereas 60.2% could imagine using online or telephone interviews as an alternative to a face-to-face meeting if regulations were changed. Only 18.7% participants reported a change in the regulations due to the current pandemic situation. CONCLUSION: Whether informed consent is obtained either online or on the telephone in the paediatric population varies widely across Europe and is not currently implemented as standard practice. For high-risk patients, such as the specific cohort of children with congenital heart defects, wider use of telemedicine might provide a benefit in the future in terms of reduced contact and reduced exposure to health risks through additional hospital stays.
RESUMEN
An elevated intracellular NADH:NAD+ ratio, or 'reductive stress', has been associated with multiple diseases, including disorders of the mitochondrial electron transport chain. As the intracellular NADH:NAD+ ratio can be in near equilibrium with the circulating lactate:pyruvate ratio, we hypothesized that reductive stress could be alleviated by oxidizing extracellular lactate to pyruvate. We engineered LOXCAT, a fusion of bacterial lactate oxidase (LOX) and catalase (CAT), which irreversibly converts lactate and oxygen to pyruvate and water. Addition of purified LOXCAT to the medium of cultured human cells with a defective electron transport chain decreased the extracellular lactate:pyruvate ratio, normalized the intracellular NADH:NAD+ ratio, upregulated glycolytic ATP production and restored cellular proliferation. In mice, tail-vein-injected LOXCAT lowered the circulating lactate:pyruvate ratio, blunted a metformin-induced rise in blood lactate:pyruvate ratio and improved NADH:NAD+ balance in the heart and brain. Our study lays the groundwork for a class of injectable therapeutic enzymes that alleviates intracellular redox imbalances by directly targeting circulating redox-coupled metabolites.
Asunto(s)
Bacterias/enzimología , Catalasa/metabolismo , Ácido Láctico/sangre , Oxigenasas de Función Mixta/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Bacterianas/metabolismo , Células HeLa , Humanos , Células K562 , NAD/metabolismo , Ácido Pirúvico/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Leigh syndrome is a devastating mitochondrial disease for which there are no proven therapies. We previously showed that breathing chronic, continuous hypoxia can prevent and even reverse neurological disease in the Ndufs4 knockout (KO) mouse model of complex I (CI) deficiency and Leigh syndrome. Here, we show that genetic activation of the hypoxia-inducible factor transcriptional program via any of four different strategies is insufficient to rescue disease. Rather, we observe an age-dependent decline in whole-body oxygen consumption. These mice exhibit brain tissue hyperoxia, which is normalized by hypoxic breathing. Alternative experimental strategies to reduce oxygen delivery, including breathing carbon monoxide (600 ppm in air) or severe anemia, can reverse neurological disease. Therefore, unused oxygen is the most likely culprit in the pathology of this disease. While pharmacologic activation of the hypoxia response is unlikely to alleviate disease in vivo, interventions that safely normalize brain tissue hyperoxia may hold therapeutic potential.
Asunto(s)
Encéfalo/metabolismo , Monóxido de Carbono/uso terapéutico , Hiperoxia/terapia , Enfermedad de Leigh/terapia , Oxígeno/metabolismo , Anemia/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Hiperoxia/metabolismo , Hipoxia/metabolismo , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Enfermedad de Leigh/metabolismo , RatonesRESUMEN
AIMS: Toll-like receptor (TLR)9 ligand CpG-oligodeoxynucleotide (CpG-ODN) exerts preconditioning in myocardial ischemia/reperfusion. We hypothesized a postconditioning effect of CpG-ODN in a murine closed-chest model of myocardial infarction. MATERIALS AND METHODS: C57BL/6 (12 weeks, male, WT) mice were instrumented at the left anterior descending artery, then allowed 5d of recovery before 30 min ischemia. Treatments comprised: 1) PBS: 250 µl phosphate buffer solution intraperitoneally 5 min before reperfusion and 2) IPC (ischemic postconditioning): 3 twenty-second reperfusion and occlusion episodes at the end of ischemia 3) CpG-ODN: 1668 thioate 0.2 µmol/kg BW intraperitoneally 5 min before reperfusion. Infarct size was assessed via triphenyltetrazolium chloride (TTC) staining after 2 and 24h reperfusion. Myocardial mRNA-expression of cytokines was measured using real-time PCR after 2h reperfusion. Phosphatidylinositol-3 kinase (PI3K)-inhibitor wortmannin was injected intraperitoneally in WT 15 min before postconditioning and PBS in each group. Cardiac function in WT was assessed with a left-ventricular pressure-volume catheter at 24h reperfusion. KEY FINDINGS: Following 30 min ischemia and 2h reperfusion, infarct size was diminished by 90% in WT postconditioned with CpG-ODN (2.4 ± 1.55 IS/AAR%) and IPC (1.98 ± 1.03 IS/AAR%) compared to PBS mice (23.2 ± 3.97 IS/AAR%). Infarct size increased following 24h reperfusion but the differences remained robust. Expression of TNF-α and IL-10 was increased in CpG-ODN. Wortmannin abolished the postconditioning effect of CpG-ODN and IPC. Ejection fraction and preload-recruitable stroke work were significantly greater in CpG-ODN mice. SIGNIFICANCE: CpG-ODN confers postconditioning via activation of TLR9. Cardiac function is preserved following CpG-ODN postconditioning. The PI3K -inhibitor wortmannin attenuates CpG-ODN postconditioning.