RESUMEN
In the isolation of polymorphonuclear neutrophils (PMNs) the technique and other external factors can have great influence on the quality and quantity of isolated neutrophils. To elucidate the influence of the blood collection technique, anticoagulants and storing temperature on isolated PMNs healthy volunteers provided blood samples with different needles and collection techniques, anticoagulants (EDTA, heparin, citrate) and storing temperatures (4, 22, 37 °C). From each blood sample PMNs were isolated and compared regarding number of PMNs and oxidative burst. The blood collection technique, anticoagulants and storing temperature had minor impact on isolated PMNs. All three tested cannulas and anticoagulants can be used to obtain blood samples for PMN isolation. For storing temperatures 37 °C should be preferred. Regarding time between the PMN isolation and the actual experiments, a time span of maximum 1 h should be targeted.
Asunto(s)
Anticoagulantes/química , Recolección de Muestras de Sangre/métodos , Separación Celular/métodos , Neutrófilos/metabolismo , Temperatura , Adulto , Donantes de Sangre , Quimiocinas/metabolismo , Citratos/química , Ácido Edético/química , Femenino , Citometría de Flujo , Voluntarios Sanos , Heparina/química , Humanos , Masculino , Agujas , Estallido Respiratorio/fisiología , Adulto JovenRESUMEN
Retrograde lung vascular perfusion can appear in high-risk surgeries. The present report is the first to study long-term retrograde perfusion of isolated perfused mouse lungs (IPLs) and to use the tyrosine kinase ephB4 and its ligand ephrinB2 as potential markers for acute lung injury. Mouse lungs were subjected to anterograde or retrograde perfusion with normal-pressure ventilation (NV) or high-pressure ventilation (=overventilation, OV) for 4 hours. Outcome parameters were cytokine, ephrinB2 and ephB4 levels in perfusate samples and bronchoalveolar lavage (BAL), and the wet-to-dry ratio. Anterograde perfusion was feasible for 4 hours, while lungs receiving retrograde perfusion presented considerable collapse rates. Retrograde perfusion resulted in an increased wet-to-dry ratio when combined with high-pressure ventilation; other physiological parameters were not affected. Cytokine levels in BAL and perfusate, as well as levels of soluble ephB4 in BAL were increased in OV, while soluble ephrinB2 BAL levels were increased in retrograde perfusion. BAL levels of ephrinB2 and ephB4 were also determined in vivo, including mice ventilated for 7 hours with normal-volume ventilation (NVV) or high-volume ventilation (HVV) with increased levels of ephB4 in HVV BAL compared to NVV. Retrograde perfusion in IPL is limited as a routine method to investigate effects due to collapse for yet unclear reasons. If successful, retrograde perfusion has an influence on pulmonary oedema formation. In BAL, ephrinB2 seems to be up-regulated by flow reversal, while ephB4 is a marker for acute lung injury.
Asunto(s)
Lesión Pulmonar Aguda/diagnóstico , Citocinas/análisis , Edema/diagnóstico , Pulmón/cirugía , Perfusión/efectos adversos , Lesión Pulmonar Aguda/inmunología , Animales , Biomarcadores/análisis , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/inmunología , Modelos Animales de Enfermedad , Edema/inmunología , Efrina-B2/análisis , Estudios de Factibilidad , Femenino , Humanos , Técnicas In Vitro/métodos , Pulmón/inmunología , Ratones , Perfusión/métodos , Receptor EphB4/análisis , Receptor EphB4/inmunología , Respiración Artificial/efectos adversos , Respiración Artificial/métodos , Factores de Tiempo , Regulación hacia ArribaRESUMEN
BACKGROUND: The acute respiratory distress syndrome (ARDS) is a serious disease in critically ill patients that is characterized by pulmonary dysfunctions, hypoxemia and significant mortality. Patients with immunodeficiency (e.g. SCID with T and B cell deficiency) are particularly susceptible to the development of severe ARDS. However, the role of T cells on pulmonary dysfunctions in immune-competent patients with ARDS is only incompletely understood. METHODS: Wild-type (wt) and RAG2-/- mice (lymphocyte deficient) received intratracheal instillations of LPS (4 mg/kg) or saline. On day 1, 4 and 10 lung mechanics and bronchial hyperresponsiveness towards acetylcholine were measured with the flexiVent ventilation set-up. The bronchoalveolar lavage fluid (BALF) was examined for leukocytes (FACS analysis) and pro-inflammatory cytokines (ELISA). RESULTS: In wt mice, lung mechanics, body weight and body temperature deteriorated in the LPS-group during the early phase (up to d4); these alterations were accompanied by increased leukocyte numbers and inflammatory cytokine levels in the BALF. During the late phase (day 10), both lung mechanics and the cell/cytokine homeostasis recovered in LPS-treated wt mice. RAG2-/- mice experienced changes in body weight, lung mechanics, BAL neutrophil numbers, BAL inflammatory cytokines levels that were comparable to wt mice. CONCLUSION: Following LPS instillation, lung mechanics deteriorate within the first 4 days and recover towards day 10. This response is not altered by the lack of T lymphocytes suggesting that T cells play only a minor role for the initiation, propagation or recovery of LPS-induced lung dysfunctions or function of T lymphocytes can be compensated by other immune cells, such as alveolar macrophages.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Citocinas/metabolismo , Síndrome de Dificultad Respiratoria/inmunología , Linfocitos T/inmunología , Lesión Pulmonar Aguda/inducido químicamente , Animales , Líquido del Lavado Bronquioalveolar/citología , Modelos Animales de Enfermedad , Femenino , Lipopolisacáridos , Pulmón/fisiopatología , Macrófagos Alveolares , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Síndrome de Dificultad Respiratoria/inducido químicamenteRESUMEN
Recently, side effects of plasma expanders like hydroxyethyl starch and gelatine gained considerable attention. Most studies have focused on the kidneys; lungs remain unconsidered. Isolated mouse lungs were perfused for 4 hours with buffer solutions based on hydroxyethyl starch (HES) 130/0.4, HES 200/0.5 or gelatine and ventilated with low or high pressure under physiological pH and alkalosis. Outcome parameters were cytokine levels and the wet-to-dry ratio. For cytokine release, murine and human PCLS were incubated in three different buffers and time points.In lungs perfused with the gelatine based buffer IL-6, MIP-2 and KC increased when ventilated with high pressure. Wet-to-dry ratios increased stronger in lungs perfused with gelatine - compared to HES 130/0.4. Alkalotic perfusion resulted in higher cytokine levels but normal wet-to-dry ratio. Murine PCLS supernatants showed increased IL-6 and KC when incubated in gelatine based buffer, whereas in human PCLS IL-8 was elevated. In murine IPL HES 130/0.4 has lung protective effects in comparison to gelatine based infusion solutions, especially in the presence of high-pressure ventilation. Gelatine perfusion resulted in increased cytokine production. Our findings suggest that gelatine based solutions may have side effects in patients with lung injury or lung oedema.