The involvement of AQP1 in heart oedema induced by global myocardial ischemia

Aquaporin‐1 (AQP1) is a member of aquaporin family that was previously proven to be involved in myocardial dysfunction; however, the role of AQP1 in myocardial stunning is less clear. To determine the change of AQP1 expression level in the heart and its effect on oedema after global myocardial ischemia, 40 adult goats underwent cardiopulmonary bypass (CPB) with an aortic cross‐clamp time of 2 h and total bypass time of 6, 12, 24, 48 and 72 h followed by subsequent reperfusion. AQP1 function of eight goats was inhibited by HgCl₂ during the 24 h on CPB. All groups were compared with eight sham bypass control goats. Myocardial water content was measured, and the APQ1 mRNA and protein levels were detected by RT‐PCR and immunoblotting, respectively. The results showed that the degree of myocardial oedema increased significantly at 6, 12, 24 and 48 h of reperfusion after CPB as compared with the control and recovered at 72 h of subsequent reperfusion. Expression levels of AQP1 mRNA and protein began to increase at 12 h and peaked at 24 h of CPB following reperfusion. Furthermore, myocardial oedema was reduced in the HgCl₂ group compared with the time‐matched CPB and control groups. These data suggested that AQP1 expression increases in CPB and AQP1 plays an important role in myocardial oedema during CPB. Copyright © 2012 John Wiley & Sons, Ltd.     

基质金属蛋白酶与体外循环肺损伤研究进展

肺泡 毛细血管基底膜损伤是体外循环 (CPB)术后肺损伤发生和发展的主要病理过程 ,基质金属蛋白酶 (MMPs)可能通过降解细胞外基质、调节细胞因子而参与CPB所致肺损伤的发生 ,研究MMPs在CPB肺损伤中的作用机制 ,对于防治CPB术后肺损伤的发生和发展具有重要意义     

Pulmonary Artery Perfusion with Anti-Tumor Necrosis Factor Alpha Antibody Reduces Cardiopulmonary Bypass-Induced Inflammatory Lung Injury in a Rabbit Model

Inflammatory lung injury is one of the main complications associated with cardiopulmonary bypass (CPB). Tumor necrosis factor-α (TNF-α) is one of the key factors mediating the CPB-induced inflammatory reactions. Our previous studies have shown that endotracheal administration of anti-tumor necrosis factor-α antibody (TNF-α Ab) produces some beneficial effects on lung in a rabbit CPB model. In this study, we further examined the effects of pulmonary artery perfusion with TNF-α Ab (27 ng/kg) on lung tissue integrity and pulmonary inflammation during CPB and investigated the mechanism underlying the TNF-α Ab-mediated effects in a rabbit model of CPB. Our results from transmission electron microscopy showed that the perfusion with TNF-α Ab alleviated CPB-induced histopathological changes in lung tissue. The perfusion with TNF-α Ab also prevented CPB-induced pulmonary edema and improved oxygenation index. Parameters indicating pulmonary inflammation, including neutrophil count and plasma TNF-α and malondialdehyde (MDA) levels, were significantly reduced during CPB by pulmonary artery perfusion with TNF-α Ab, suggesting that the perfusion with TNF-α Ab reduces CPB-induced pulmonary inflammation. We further investigated the molecular mechanism underlying the protective effects of TNF-α Ab on lung. Our quantitative RT-PCR analysis revealed that pulmonary artery perfusion with TNF-α Ab significantly decreased TNF-α expression in lung tissue during CPB. The apoptotic index in lung tissue and the expression of proteins that play stimulatory roles in apoptosis pathways including the fas ligand (FasL) and Bax were markedly reduced during CPB by the perfusion with TNF-α Ab. In contrast, the expression of Bcl-2, which plays an inhibitory role in apoptosis pathways, was significantly increased during CPB by the perfusion with TNF-α Ab, indicating that the perfusion with TNF-α Ab significantly reduces CPB-induced apoptosis in lung. Thus, our study suggests that pulmonary artery perfusion with TNF-α Ab might be a promising approach for attenuating CPB-induced inflammatory lung injury.     

Pre-emptive and continuous inhaled NO counteracts the cardiopulmonary consequences of extracorporeal circulation in a pig model.

Cardiopulmonary bypass (CPB) activates a systemic inflammatory response characterized clinically by alterations in cardiovascular and pulmonary function. The aim of this study was to measure the cardiopulmonary consequences in sham-operated pigs, and in animals subjected to CPB in the presence or absence of lipopolysaccharide (LPS). We also investigated, if the perioperative administration of inhaled NO exerts significant cardiopulmonary effects in an anaesthetized and mechanically ventilated pig model of extracorporeal circulation. Thirty pigs were randomized into six equal groups (sham; sham+INO; CPB; CPB+INO; CPB+LPS; CPB+LPS+INO) and subjected to anaesthesia with mechanical ventilation for up to 24h. We found that CPB+LPS group has the highest degree of lung injury. We also demonstrated that there was a significant difference on the cardiovascular parameters (heart rate, central venous pressure, stroke volume index, and mean systemic arterial blood pressure) between the CPB groups and the sham groups. The deteriorated lung mechanics was associated with a decrease in active subfraction of surfactant (LA) with time during the procedure (P=0.0003), on which inhaled NO had only an initial beneficial effect. In our model, inhaled NO had no long-term beneficial effect on lung mechanics and surfactant homeostasis despite improving lung haemodynamics, inflammation, and oxygenation. We conclude from this study that the use of pre-emptive and continuous inhaled NO therapy has protective and safe effects against lung ischemia/reperfusion associated with CPB.     

Intervention of rosiglitazone on myocardium Glut-4 mRNA expression during ischemia–reperfusion injury in cardio-pulmonary bypass in dogs

During cardiac pulmonary bypass (CPB), myocardial ischemia–reperfusion (I/R) induces heart glucose metabolism impairment. Our previous research showed that the decreased glucose utilization is due to decreased glucose transporter-4 (Glut-4) expression and translocation to myocyte surface membranes. This study further examined whether rosiglitazone, a synthetic agonist of peroxisome proliferator-activated receptor γ, could intervene glucose metabolism by regulating Glut-4 mRNA during I/R in dogs. Cardiac ischemia was induced by cardiopulmonary bypass for 30 or 120 min. Plasma insulin and glucose concentrations were measured at pre-bypass (control), aortic cross-clamp off (I/R) at 15, 45, and 75 min. The left ventricle biopsies were taken for the expression of Glut-4 mRNA by real-time RT-PCR. In dogs receiving 120 min ischemia, coronary arterial, venous glucose concentrations, plasma insulin levels, and insulin resistant index (IRI) were increased, but the expression of Glut-4 mRNA was decreased obviously at 15 min of reperfusion, and recovered gradually. On the other hand, these changes were relatively mild in dogs treated with rosiglitazone in cardioplegic solution and expression of Glut-4 mRNA was increased remarkably. It is concluded that the decrease in total amount of Glut-4 mRNA expression could be one of the important molecular mechanisms, which causes the myocardium insulin resistance. The longer the ischemia period, the decrease in amount of Glut-4 mRNA was more dramatic. Adding rosiglitazone into the cardioplegic solution during I/R can increase the amount of Glut-4 mRNA expression, mitigate the myocardium insulin resistance and improve the myocardium I/R injury during CPB.     

Whole body protection during three hours of total circulatory arrest: An experimental study

Survival following 3 hr of total circulatory arrest under profound hypothermic conditions was explored in 19 adult mongrel dogs. Thermoregulatory management included combined surface/perfusion hypothermia and azeotrope anesthesia in 95% O2/5% CO2. All animals were resuscitated and survived for at least 12 hr. During the last seven trials (Group II) the following principles were applied: uniform whole-body cooling where differences between rectal, esophageal, and pharyngeal temperatures averaged less than 1 degree C, induction of circulatory arrest at approximately 3 degrees C, constant lung inflation (10-12 cm H2O between 20 degrees C cooling and 20 degrees C rewarming, including the 3-hr arrest period) and ventilation assistance with positive end-expiratory pressure (4 cm H2O) after 20 degrees C rewarming, intraoperative maintenance of colloid osmotic pressure (COP) above 11 mm Hg, replacement of the cooling perfusate with a colloid-rich rewarming prime (COP = 15 mm Hg) and restoration of hemostasis with fresh whole blood transfusions. The application of these principles resulted in the long-term survival of five animals with four survivors displaying no clinically detectable neurological abnormalities. However, two animals developed optic impairment and one animal died from intusseption on the fourth postoperative day. Despite the improved results, it should also be noted that during pilot (Group I) studies (from which the aforementioned principles were derived) fatalities from complications attributed to systemic edema, central nervous system, or pulmonary or coagulation dysfunctions occurred in 9 out of 12 trials. We conclude that whole body protection following 3 hr of total circulatory arrest at a uniform temperature less than 5 degrees C can be successfully accomplished.     

Effect of ischemia and reperfusion without airway occlusion on vascular barrier function in the in vivo mouse lung.

Ischemia-reperfusion (I/R) lung injury causes increased vascular permeability and edema. We developed an in vivo murine model of I/R allowing measurement of pulmonary vascular barrier function without airway occlusion. The left pulmonary artery (PA) was occluded with an exteriorized, slipknotted suture in anesthetized C57BL/6J mice. The effect of ischemic time was determined by subjecting mice to 5, 10, or 30 min of left lung ischemia followed by 150 min of reperfusion. The effect of reperfusion time was determined by subjecting mice to 30 min of left lung ischemia followed by 30 or 150 min of reperfusion. Changes in pulmonary vascular barrier function were measured with the Evans blue dye (EBD) technique, dual-isotope radiolabeled albumin (RA), bronchoalveolar lavage (BAL) protein concentration, and wet weight-to-dry weight ratio (WW/DW). Increasing left lung ischemia with constant reperfusion time or increasing left lung reperfusion time after constant ischemic time resulted in significant increases in left lung EBD content at all times compared with both right lung values and sham surgery mice. The effects of left lung ischemia on lung EBD were corroborated by RA but the effects of increasing reperfusion time differed, suggesting binding of EBD to lung tissue. An increase in WW/DW was only detected after 30 min of reperfusion, suggesting edema clearance. BAL protein concentrations were unaffected. We conclude that short periods of I/R, without airway occlusion, increase pulmonary vascular permeability in the in vivo mouse, providing a useful model to study molecular mechanisms of I/R lung injury.     

Effect of bronchial artery blood flow on cardiopulmonary bypass-induced lung injury

Cardiovascular surgery requiring cardiopulmonary bypass (CPB) is frequently complicated by postoperative lung injury. Bronchial artery (BA) blood flow has been hypothesized to attenuate this injury. The purpose of the present study was to determine the effect of BA blood flow on CPB-induced lung injury in anesthetized pigs. In eight pigs (BA ligated) the BA was ligated, whereas in six pigs (BA patent) the BA was identified but left intact. Warm (37 degrees C) CPB was then performed in all pigs with complete occlusion of the pulmonary artery and deflated lungs to maximize lung injury. BA ligation significantly exacerbated nearly all aspects of pulmonary function beginning at 5 min post-CPB. At 25 min, BA-ligated pigs had a lower arterial Po(2) at a fraction of inspired oxygen of 1.0 (52 +/- 5 vs. 312 +/- 58 mmHg) and greater peak tracheal pressure (39 +/- 6 vs. 15 +/- 4 mmHg), pulmonary vascular resistance (11 +/- 1 vs. 6 +/- 1 mmHg x l(-1) x min), plasma TNF-alpha (1.2 +/- 0.60 vs. 0.59 +/- 0.092 ng/ml), extravascular lung water (11.7 +/- 1.2 vs. 7.7 +/- 0.5 ml/g blood-free dry weight), and pulmonary vascular protein permeability, as assessed by a decreased reflection coefficient for albumin (sigma(alb); 0.53 +/- 0.1 vs. 0.82 +/- 0.05). There was a negative correlation (R = 0.95, P < 0.001) between sigma(alb) and the 25-min plasma TNF-alpha concentration. These results suggest that a severe decrease in BA blood flow during and after warm CPB causes increased pulmonary vascular permeability, edema formation, cytokine production, and severe arterial hypoxemia secondary to intrapulmonary shunt.     

Transient Th1/Th2 disbalance indicates postoperative effusions and edema after cardiopulmonary bypass in children.

BACKGROUND: Cardiac surgery with cardiopulmonary bypass (CPB) induces substantial release of IL-10, indicating increased Th2 cell response. Therefore, in this study, we wanted to verify if this response is due to CPB or surgical trauma, and to study its relation to postoperative effusions and edema (POEE) in children. METHODS: Th1/Th2 reaction was monitored in children undergoing cardiovascular surgery with (n = 75) and without CPB (n = 29). RESULTS: Surgery with CPB compared to surgery without CPB induced a transient shift towards Th2. Elevated Th2 response was related to increased vascular permeability and POEE. CONCLUSION: The immune suppression/Th2 response is typical for CPB, and at intermediate level is tolerable but at high level could be adverse for the patients.     

川芎嗪对家兔肺缺血/再灌注损伤时Fas/FasL基因表达的影响

目的:探讨川芎嗪对肺缺血/再灌注损伤(PI/RI)时Fas/FasL基因表达的影响。方法:采用在体兔单肺原位缺血-再灌注模型。实验兔90只,随机均分为假手术对照组(Sham)、肺缺血/再灌注组(I/R)和肺缺血/再灌注加川芎嗪治疗组(LGT)。每组又分为再灌注1h3、h、5 h三个亚组,每个亚组10只,分别于再灌注1 h3、h、5 h三个时点取左肺组织,观察Fas/Fas配体(Fas/FasL)mRNA定位表达、凋亡指数(AI)、肺组织湿、干重比(W/D)、肺损伤组织学定量评价指标(IQA)及光镜、电镜下的组织形态学改变。结果:肺再灌注1 h、3 h、5 h,LGT组Fas/FasLmRNA在肺小动脉内(外)膜、肺小静脉内膜、肺泡上皮及肺支气管上皮弱阳性表达,与I/R组同一时点比较阳性表达明显减弱;AI、W/D和IQA值显著低于I/R组(P<0.01和P<0.05);肺组织形态学异常改变不同程度减轻。结论:川芎嗪可下调肺组织Fas/FasL mRNA的表达而减轻细胞凋亡,对PI/RI发挥积极的防护作用。     

Effect of NADPH oxidase inhibition on cardiopulmonary bypass-induced lung injury

Cardiopulmonary bypass (CPB) causes acute lung injury. Reactive oxygen species (ROS) from NADPH oxidase may contribute to this injury. To determine the role of NADPH oxidase, we pretreated pigs with structurally dissimilar NADPH oxidase inhibitors. Low-dose apocynin (4-hydroxy-3-methoxy-acetophenone; 200 mg/kg, n = 6), high-dose apocynin (400 mg/kg, n = 6), or diphenyleneiodonium (DPI; 8 mg/kg) was compared with diluent (n = 8). An additional group was treated with indomethacin (10 mg/kg, n = 3). CPB was performed for 2 h with deflated lungs, complete pulmonary artery occlusion, and bronchial artery ligation to maximize lung injury. Parameters of pulmonary function were evaluated for 25 min following CPB. Blood chemiluminescence indicated neutrophil ROS production. Electron paramagnetic resonance determined the effect of apocynin and DPI on in vitro pulmonary endothelial ROS production following hypoxia-reoxygenation. Both apocynin and DPI attenuated blood chemiluminescence and post-CPB hypoxemia. At 25 min post-CPB with Fi(O(2)) = 1, arterial Po(2) (Pa(o(2))) averaged 52 +/- 5, 162 +/- 54, 335 +/- 88, and 329 +/- 119 mmHg in control, low-dose apocynin, high-dose apocynin, and DPI-treated groups, respectively (P < 0.01). Indomethacin had no effect. Pa(O(2)) correlated with blood chemiluminescence measured after drug administration before CPB (R = -0.60, P < 0.005). Neither apocynin nor DPI prevented the increased tracheal pressure, plasma cytokine concentrations (tumor necrosis factor-alpha and IL-6), extravascular lung water, and pulmonary vascular protein permeability observed in control pigs. NADPH oxidase inhibition, but not xanthine oxidase inhibition, significantly blocked endothelial ROS generation following hypoxia-reoxygenation (P < 0.05). NADPH oxidase-derived ROS contribute to the severe hypoxemia but not to the increased cytokine generation and pulmonary vascular protein permeability, which occur following CPB.     

Low degree of activation of circulating neutrophils determined by flow cytometry during cardiac surgery with cardiopulmonary bypass

BACKGROUND: Enhanced expression of adhesion molecules LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) following cardiac surgery with cardiopulmonary bypass (CPB) is held responsible for postoperative complications. Surface expression of these molecules, intracellular pH (pH(i)), and oxidative burst capacity was analyzed to test for neutrophil activation during pediatric cardiac surgery. METHODS: Blood samples were drawn from 36 patients (age: 3--16 years) 24 h preoperatively, after onset of anesthesia, after connection to CPB (CPB1, before and after passing CPB, n = 15), at reperfusion (CPB2), and up to 7 days postoperatively. Cells adhering to CPB filters were isolated (n = 11). Antigen expression, pH(i), and oxidative burst capacity on neutrophils was analyzed by flow cytometry. RESULTS: During surgery, oxidative burst capacity was at low level with a mild increase only 1 day after surgery. pH(i) was decreased throughout the surgery. Surgery induced more than 36% decrease of LFA-1 and Mac-1 expression (P < 0.03). Up to postoperative day 7, no increase of antigen expression above baseline was found. Neutrophils isolated from filters of the CPB had increased LFA-1 and Mac-1 expression (all P < 0.05). Integrin expression on neutrophils passing the CPB at CPB1 was decreased (P < 0.05). CONCLUSION: Reduced adhesion molecule expression on neutrophils may be due to selective filtration of highly adhesive cells. This, in combination with low-level oxidative burst capacity, induced by immunosuppressive cytokines (e.g., interleukin-10), reduced the neutrophil activity. Our data indicate that increased activity of circulating neutrophils cannot exclusively be held responsible for postoperative complications after surgery with CPB.     

Poly(ADP-ribose) polymerase inhibitor PJ-34 reduces mesenteric vascular injury induced by experimental cardiopulmonary bypass with cardiac arrest

The aim of this study was to investigate effects of poly(ADP-ribose) polymerase (PARP) inhibition on mesenteric vascular function and metabolism in an experimental model of cardiopulmonary bypass (CPB) with cardiac arrest. Twelve anesthetized dogs underwent 90-min hypothermic CPB. After 60 min of cardiac arrest, reperfusion was started for 40 min following application of either saline vehicle (control, n = 6) or a potent PARP inhibitor, PJ-34 (10 mg/kg iv bolus and 0.5 mg.kg(-1).min(-1) infusion for 20 min, n = 6). PJ-34 led to better recovery of cardiac output (2.2 +/- 0.1 vs. 1.8 +/- 0.2 l/min in control) and mesenteric blood flow (175 +/- 38 vs. 83 +/- 4 ml/min, P < 0.05 vs. control) after reperfusion. The impaired vasodilator response of the superior mesenteric artery to acetylcholine, assessed in the control group after CPB (-32.8 +/- 3.3 vs. -57.6 +/- 6.6% at baseline, P < 0.05), was improved by PJ-34 (-50.3 +/- 3.6 vs. -54.3 +/- 4.1% at baseline, P < 0.05 vs. control). Although plasma nitrate/nitrite concentrations were not significantly different between groups, mesenteric nitric oxide synthase activity was increased in the PJ-34 group (P < 0.05). Moreover, the treated group showed a marked attenuation of mesenteric venous plasma myeloperoxidase levels after CPB compared with the control group (75 +/- 1 vs. 135 +/- 9 ng/ml, P < 0.05). Pharmacological PARP inhibition protects against development of post-CPB mesenteric vascular dysfunction by improving hemodynamics, restoring nitric oxide production, and reducing neutrophil adhesion.     

Bronchial circulation in pulmonary artery occlusion and reperfusion

Obstruction of pulmonary arterial blood flow results in minimal biochemical and/or morphological changes in the involved lung. If the lung is reperfused, a syndrome of leukopenia and lung edema occurs. We used the radiolabeled microsphere technique to measure the response of the bronchial circulation in rabbits to acute pulmonary artery occlusion (PAO) and to pulmonary artery reperfusion. We found that the bronchial blood flow (Qbr) decreased from a base line of 0.37 +/- 0.10 to 0.09 +/- 0.04 (SE) ml.min-1.g dry lung-1 (P less than or equal to 0.05) after 4 h of PAO. In a separate group of animals, Qbr 24 h after PAO remained low (0.20 +/- 0.07 ml.min-1.g dry lung-1, P = 0.06). Qbr during PAO was inversely correlated with the wet-to-dry ratio after reperfusion (r = -0.68, P = 0.06). Qbr did not change during 4 h of reperfusion. We speculate that a critical level of Qbr may be necessary during PAO to prevent ischemia/reperfusion injury from occurring.     

Examination of Physiological Function and Biochemical Disorders in a Rat Model of Prolonged Asphyxia-Induced Cardiac Arrest followed by Cardio Pulmonary Bypass Resuscitation

Background

Cardiac arrest induces whole body ischemia, which causes damage to multiple organs particularly the heart and the brain. There is clinical and preclinical evidence that neurological injury is responsible for high mortality and morbidity of patients even after successful cardiopulmonary resuscitation. A better understanding of the metabolic alterations in the brain during ischemia will enable the development of better targeted resuscitation protocols that repair the ischemic damage and minimize the additional damage caused by reperfusion.

Method

A validated whole body model of rodent arrest followed by resuscitation was utilized; animals were randomized into three groups: control, 30 minute asphyxial arrest, or 30 minutes asphyxial arrest followed by 60 min cardiopulmonary bypass (CPB) resuscitation. Blood gases and hemodynamics were monitored during the procedures. An untargeted metabolic survey of heart and brain tissues following cardiac arrest and after CPB resuscitation was conducted to better define the alterations associated with each condition.

Results

After 30 min cardiac arrest and 60 min CPB, the rats exhibited no observable brain function and weakened heart function in a physiological assessment. Heart and brain tissues harvested following 30 min ischemia had significant changes in the concentration of metabolites in lipid and carbohydrate metabolism. In addition, the brain had increased lysophospholipid content. CPB resuscitation significantly normalized metabolite concentrations in the heart tissue, but not in the brain tissue.

Conclusion

The observation that metabolic alterations are seen primarily during cardiac arrest suggests that the events of ischemia are the major cause of neurological damage in our rat model of asphyxia-CPB resuscitation. Impaired glycolysis and increased lysophospholipids observed only in the brain suggest that altered energy metabolism and phospholipid degradation may be a central mechanism in unresuscitatable brain damage.     

Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.     

细胞自噬在大鼠肺缺血/再灌注引发肝脏损伤中的作用*

目的: 探讨肺缺血/再灌注(LI/R)时肝脏损伤的影响,并初步探索细胞自噬(Autophagy)在其中发挥的作用。方法: 构建大鼠缺血/再灌注肺损伤(LI/RI)模型,模型制备方法为大鼠麻醉后切开气管进行机械通气,使用动脉夹将肺门夹闭模拟缺血过程,30 min后松开动脉夹,恢复灌注3 h。24只大鼠随机分为伪手术组(Sham组)、缺血/再灌注组(I/R组)、溶剂组(DMSO组)和自噬抑制剂组(3-MA组),每组均6只,后2组大鼠术前分别腹腔注射DMSO和3-MA,造模结束后使用肺湿/干重比判断造模是否成功;抽取静脉血测定肝脏转氨酶指标ALT与AST;取肝脏组织,光镜下观察肝脏形态改变,以及电镜下观察肝细胞超微结构;使用RT-qPCR和Western blot实验分别检测肝脏组织细胞中自噬相关蛋白的基因mRNA表达水平和蛋白表达水平。结果: 与Sham组相比,其余各组肺湿/干重比均升高;血AST和ALT均有大幅升高且肝脏组织损伤明显,其中以I/R组升高最为明显,光镜下组织形态学及电镜下细胞微细结构均有不同程度的破坏;肝脏中自噬相关蛋白的基因表达水平与蛋白表达水平均有明显不同,表现为自噬上升 (P＜0.01或P＜0.05)。I/R组和DMSO组肝脏组织均有较重损伤,肝细胞结构破坏严重,自噬小体形成,而AST、ALT、自噬相关蛋白转录和表达水平等各项指标均无统计学差异(P＞0.05)。而相较于DMSO组,3-MA组肝脏组织损伤有所减轻,肝细胞微细结构损伤程度低,且无自噬小体形成,血中AST和ALT下降,肝脏组织内自噬水平均下降 (P＜0.05)。结论: 肺缺血/再灌注可引起大鼠肝损伤;细胞自噬可介导大鼠肺缺血/再灌注引起的肝损伤,抑制细胞自噬可以有效减轻大鼠LI/R引起的肝损伤。     

Vagal afferents and active upper airway closure during pulmonary edema in lambs.

The present study was undertaken to gain further insight into the mechanisms responsible for the sustained active expiratory upper airway closure previously observed during high-permeability pulmonary edema in lambs. The experiments were conducted in nonsedated lambs, in which airflow and thyroarytenoid and inferior pharyngeal constrictor muscle electromyographic activity were recorded. We first studied the consequences of hemodynamic pulmonary edema (induced by impeding pulmonary venous return) on upper airway dynamics in five lambs; under this condition, a sustained expiratory upper airway closure consistently appeared. We then tested whether expiratory upper airway closure was related to vagal afferent activity from bronchopulmonary receptors. Five bivagotomized lambs underwent high-permeability pulmonary edema: no sustained expiratory upper airway closure was observed. Finally, we studied whether a sustained decrease in lung volume induced a sustained expiratory upper airway closure. Five lambs underwent a 250-ml pleural infusion: no sustained expiratory upper airway closure was observed. We conclude that 1) the sustained expiratory upper airway closure observed during pulmonary edema in nonsedated lambs is related to stimulation of vagal afferents by an increase in lung water and 2) a decrease in lung volume does not seem to be the causal factor.     

Protection from pulmonary ischemia-reperfusion injury by adenosine A2A receptor activation

Background

Lung ischemia-reperfusion (IR) injury leads to significant morbidity and mortality which remains a major obstacle after lung transplantation. However, the role of various subset(s) of lung cell populations in the pathogenesis of lung IR injury and the mechanisms of cellular protection remain to be elucidated. In the present study, we investigated the effects of adenosine A_2A receptor (A_2AAR) activation on resident lung cells after IR injury using an isolated, buffer-perfused murine lung model.

Methods

To assess the protective effects of A_2AAR activation, three groups of C57BL/6J mice were studied: a sham group (perfused for 2 hr with no ischemia), an IR group (1 hr ischemia + 1 hr reperfusion) and an IR+ATL313 group where ATL313, a specific A_2AAR agonist, was included in the reperfusion buffer after ischemia. Lung injury parameters and pulmonary function studies were also performed after IR injury in A_2AAR knockout mice, with or without ATL313 pretreatment. Lung function was assessed using a buffer-perfused isolated lung system. Lung injury was measured by assessing lung edema, vascular permeability, cytokine/chemokine activation and myeloperoxidase levels in the bronchoalveolar fluid.

Results

After IR, lungs from C57BL/6J wild-type mice displayed significant dysfunction (increased airway resistance, pulmonary artery pressure and decreased pulmonary compliance) and significant injury (increased vascular permeability and edema). Lung injury and dysfunction after IR were significantly attenuated by ATL313 treatment. Significant induction of TNF-α, KC (CXCL1), MIP-2 (CXCL2) and RANTES (CCL5) occurred after IR which was also attenuated by ATL313 treatment. Lungs from A_2AAR knockout mice also displayed significant dysfunction, injury and cytokine/chemokine production after IR, but ATL313 had no effect in these mice.

Conclusion

Specific activation of A_2AARs provides potent protection against lung IR injury via attenuation of inflammation. This protection occurs in the absence of circulating blood thereby indicating a protective role of A_2AAR activation on resident lung cells such as alveolar macrophages. Specific A_2AAR activation may be a promising therapeutic target for the prevention or treatment of pulmonary graft dysfunction in transplant patients.