Lung reperfusion in dogs causes bilateral lung injury

Occlusion of the pulmonary arterial circulation to a lung for prolonged periods has been reported to result in only minimal alterations in lung morphology. We studied the effects of 48 h of pulmonary arterial occlusion followed by 4 h of reperfusion in 18 awake dogs. Because of evidence in other organ systems of O2 radical generation, during reperfusion, nine of the animals were randomly assigned to receive allopurinol, a xanthine oxidase inhibitor, and vitamin E, an antioxidant. Reperfusion resulted in marked edema and inflammatory infiltrates in the reperfused lung but also caused mild edema and inflammation in the contralateral continuously perfused lung. Electron microscopy demonstrated lysis of both capillary endothelial and alveolar epithelial cells bilaterally, with the frequency of cell injury greater on the reperfused side. During reperfusion, body temperatures rose dramatically from 39.4 +/- 0.1 to 40.6 +/- 0.2 degrees C (P less than 0.05) and marked leukopenia developed. There were no differences in any hemodynamic, gas exchange, or morphometric measurements between allopurinol-treated dogs and untreated animals. We conclude that reperfusion causes local and distant injury which does not appear to be mediated by xanthine oxidase-produced O2 radicals.     

Thromboxane does not mediate pulmonary hypertension in phorbol ester-induced acute lung injury in dogs

Thromboxane (Tx) has been suggested to mediate the pulmonary hypertension of phorbol myristate acetate- (PMA) induced acute lung injury. To test this hypothesis, the relationship between Tx and pulmonary arterial pressure was evaluated in a model of acute lung injury induced with PMA in pentobarbital sodium-anesthetized male mongrel dogs. Sixty minutes after administration of PMA (20 micrograms/kg iv, n = 10), TxB2 increased 10-fold from control in both systemic and pulmonary arterial blood and 8-fold in bronchoalveolar lavage (BAL) fluid. Concomitantly, pulmonary arterial pressure (Ppa) increased from 14.5 +/- 1.0 to 36.2 +/- 3.5 mmHg, and pulmonary vascular resistance (PVR) increased from 5.1 +/- 0.4 to 25.9 +/- 2.9 mmHg.l-1.min. Inhibition of Tx synthase with OKY-046 (10 mg/kg iv, n = 6) prevented the PMA-induced increase in Tx concentrations in blood and BAL fluid but did not prevent or attenuate the increase in Ppa. OKY-046 pretreatment did, however, attenuate but not prevent the increase in PVR 60 min after PMA administration. Pretreatment with the TxA2/prostaglandin H2 receptor antagonist ONO-3708 (10 micrograms.kg-1.min-1 iv, n = 7) prevented the pressor response to bolus injections of 1-10 micrograms U-46619, a Tx receptor agonist, but did not prevent or attenuate the PMA-induced increase in Ppa. ONO-3708 also attenuated but did not prevent the increase in PVR. These results suggest that Tx does not mediate the PMA-induced pulmonary hypertension but may augment the increases in PVR in this model of acute lung injury.     

Halofuginone does not reduce fibrosis in bleomycin-induced lung injury

Halofuginone, a coccidiostatic alkaloid, has anti-fibrotic properties, and may be useful as a therapeutic agent in lung fibrosis. To test this hypothesis we investigated the effect of halofuginone on bleomycin-induced lung fibrosis in Sprague-Dawley rats. Treatment groups included: (1) a single intratracheal (IT) instillation of 1.2U bleomycin, and intraperitoneal (IP) injection of halofuginone (0.5 mg/dose), every other day; (2) IT 1.2U bleomycin and IP distilled water (D.W.), every other day; (3) IT 0.8U bleomycin and daily IP halofuginone (0.5 mg/dose); (4) IT 0.8U bleomycin and daily IP D.W.; (5) IT saline and IP halofuginone, every other day; (6) IT saline and daily IP D.W.; (7) IT 0.625U bleomycin and oral halofuginone (10 mg/kg rodent lab chow); (8) IT 0.625U bleomycin and standard lab chow. Animals were studied 14 days after IT instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage fluid, by a semi-quantitative morphological index of lung injury, and by biochemical analysis of lung hydroxyproline content. Overt signs of lung injury were apparent in bleomycin-treated rats by all measures. These changes were not affected by treatment with halofuginone, irrespective of the treatment regimen used. This study does not support the use of halofuginone to prevent or ameliorate lung fibrosis.     

Tumor necrosis factor-alpha does not cause lung edema in rabbits.

Although tumor necrosis factor-alpha (TNF) is a key mediator in the pathophysiology of sepsis and septic shock, its role in lung microvascular injury is controversial. In isolated blood-perfused rabbit lungs, we studied the microvascular effects of human recombinant TNF by measuring the capillary filtration coefficient (Kf,c) as an index of microvascular leakiness and the arterial and venous resistances and occlusion pressures to define the microvascular pressure profile. At the end of the experiments, the lung wet-to-dry weight ratio (W/D) was determined as an index of edema. TNF increased the pulmonary venous resistance slightly but did not affect Kf,c or W/D. Furthermore, TNF at different doses failed to increase W/D less than or equal to 8 h after in vivo administration. Our data suggest that 1) the pulmonary microvascular response to TNF differs from the systemic response, which is characterized by arteriolar vasodilation, and 2) TNF is insufficient to cause lung edema, both in vivo and in vitro. Thus the development of lung microvascular injury may require the combined action of TNF and other mediators.     

红花对兔肺缺血/再灌注损伤及环氧酶表达的影响

目的：探讨红花（safflor injection,SI）抗肺缺血/再灌注损伤作用及其机制。方法：复制在体兔肺缺血/再灌注损伤模型。30只日本大耳兔,随机均分为三组：假手术组（S组）,缺血/再灌注组（I/R组）和缺血/再灌注＋红花注射液组（SI组）。实验结束时,自颈动脉抽血检测丙二醛（MDA）含量、超氧化物歧化酶（SOD）和黄嘌呤氧化酶（XO）活力。取肺组织测湿干重比（W/D）,计算肺泡损伤率（IAR）,电镜观察细胞超微结构改变。免疫组化法检测肺组织COX-1、COX-2蛋白表达的变化 组织原位杂交法检测肺组织COX-1mRNA、COX-2mRNA表达的变化。结果：I/R组血清MDA、XO均显著高于S组,SOD明显低于S组（P〈0.01） I/R组和SI组的W/D与IAR均高于S组（均P〈0.05和P〈0.01）,SI组显著低于I/R组（P〈0.01） I/R组肺组织的超微结构损伤严重,SI组损伤程度明显较轻 免疫组化和原位杂交发现I/R组肺组织COX-2蛋白和COX-2mRNA表达皆显著高于SI组（均P〈0.01）,肺组织COX-1蛋白和COX-1mRNA表达三组间无明显变化。结论：肺缺血/再灌注损伤可诱导肺组织COX-2的表达,SI可能通过抗氧化应激和下调肺组织COX-2蛋白及其基因的表达而减轻肺缺血/再灌注损伤。     

Lack of alveolar O2 during lung reperfusion does not decrease edema formation

We previously reported that pulmonary arterial occlusion for 48 h followed by 4 h of reperfusion in awake dogs results in marked edema and inflammatory infiltrates in both reperfused and contralateral lungs (Am. Rev. Respir. Dis. 134: 752-756, 1986; J. Appl. Physiol. 63: 942-950, 1987). In this experiment we study the effects of alveolar hypoxia on this injury. Anesthetized dogs underwent thoracotomy and occlusion of the left pulmonary artery. Twenty-four hours later the dogs were reanesthetized, and a double-lumen endotracheal tube was placed. The right lung was continuously ventilated with an inspiratory O2 fraction (FIO2) of 0.35. In seven study animals the left lung was ventilated with an FIO2 of 0 for 3 h after the left pulmonary artery occluder was removed. In six control animals the left lung was ventilated with an FIO2 of 0.35 during the same reperfusion period. Postmortem bloodless wet-to-dry weight ratios were 5.87 +/- 0.20 for the left lower lobe and 5.32 +/- 0.12 for the right lower lobe in the dogs with hypoxic ventilation (P less than 0.05 for right vs. left lobes). These values were not significantly different from the control dog lung values of 5.94 +/- 0.22 for the left lower lobe and 5.11 +/- 0.07 for the right lower lobe (P less than 0.05 for right vs. left lobes). All values were significantly higher than our laboratory normal of 4.71 +/- 0.06. We conclude that reperfusion injury is unaffected by alveolar hypoxia during the reperfusion phase.     

Dexamethasone does not ameliorate gliosis in a mouse model of neurodegenerative disease

Prolonged neuroinflammation is a driving force for neurodegenerative disease, and agents against inflammatory responses are regarded as potential treatment strategies. Here we aimed to evaluate the prevention effects on gliosis by dexamethasone (DEX), an anti-inflammation drug. We used DEX to treat the nicastrin conditional knockout (cKO) mouse, a neurodegenerative mouse model. DEX (10 mg/kg) was given to 2.5-month-old nicastrin cKO mice, which have not started to display neurodegeneration and gliosis, for 2 months. Immunohistochemistry (IHC) and Western blotting techniques were used to detect changes in neuroinflammatory responses. We found that activation of glial fibrillary acidic protein (GFAP) positive or ionized calcium binding adapter molecule1 (Iba1) positive cells was not inhibited in nicastrin cKO mice treated with DEX as compared to those treated with saline. These data suggest that DEX does not prevent or ameliorate gliosis in a neurodegenerative mouse model when given prior to neuronal or synaptic loss.     

Pentoxifylline does not attenuate acute lung injury in the absence of granulocytes.

Pentoxifylline (PTX), a methylxanthine, can suppress polymorphonuclear leukocyte (PMN) activation and attenuate sepsis-induced acute lung injury. We investigated whether PTX prevents non-PMN-dependent lung injury. First we studied four groups of granulocyte-depleted guinea pigs (control, PTX, Escherichia coli, and E. coli + PTX). Lung injury was assessed by wet-to-dry lung weight (W/D) ratio and lung tissue-to-plasma 125I-albumin ratio (albumin index, AI). The E. coli group showed a significant increase in the lung W/D ratio and AI compared with the control and PTX groups. However, PTX did not prevent the E. coli-induced increase in the lung W/D ratio and AI. Next we investigated the effects of PTX on endothelial cell monolayer permeability and adenosine 3',5'-cyclic monophosphate (cAMP) levels. Whereas E. coli lipopolysaccharide (LPS) alone increased the endothelial permeability, PMNs added to the endothelial monolayers and exposed to LPS enhanced the increase. PTX attenuated the permeability increase mediated by LPS-exposed PMNs. PTX did not prevent the LPS-induced increase in permeability when PMNs were not present, although PTX increased endothelial cell cAMP levels. These data demonstrate that 1) PTX does not prevent lung injury in granulocyte-depleted guinea pigs; 2) PTX does not prevent LPS-induced increases in endothelial cell permeability, despite increased cAMP levels; and 3) PTX attenuates PMN-dependent increases in endothelial cell permeability.     

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.     

Monokine-induced acute lung injury in rabbits

Interleukin-1 (IL-1) mediates components of the acute phase response, stimulates granulocyte metabolism, and induces endothelial cell surface changes. We studied in unanesthetized rabbits the effects of intravenous divided dose infusions of a murine monokine preparation containing IL-1 activity, on circulating granulocytes, their sequestration within the pulmonary microvasculature, pulmonary edema formation, and changes in pulmonary vascular permeability. Monokine administration induced significant (P less than 0.01) granulocytopenia as well as a significant (P less than 0.001) increase in mean alveolar septal wall granulocytes per high power field (HPF) compared with saline-injected controls. Infusions of the monokine preparation significantly (P less than 0.005) increased lung wet-to-dry weight ratios as well as significantly (P less than 0.025) increased pulmonary extravasation of radiolabeled albumin. Electron microscopic analysis of lung sections obtained from monokine-infused animals demonstrated endothelial injury, perivascular edema, and extravasation of an ultrastructural tracer. We conclude that a monokine preparation containing IL-1 activity can induce profound granulocytopenia, pulmonary leukostasis, and acute pulmonary vascular endothelial injury.     

Angiopoietin-1 treatment reduces inflammation but does not prevent ventilator-induced lung injury

Background

Loss of integrity of the epithelial and endothelial barriers is thought to be a prominent feature of ventilator-induced lung injury (VILI). Based on its function in vascular integrity, we hypothesize that the angiopoietin (Ang)-Tie2 system plays a role in the development of VILI. The present study was designed to examine the effects of mechanical ventilation on the Ang-Tie2 system in lung tissue. Moreover, we evaluated whether treatment with Ang-1, a Tie2 receptor agonist, protects against inflammation, vascular leakage and impaired gas exchange induced by mechanical ventilation.

Methods

Mice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with either an inspiratory pressure of 10 cmH₂O (‘low’ tidal volume ∼7.5 ml/kg; LV_T) or 18 cmH₂O (‘high’ tidal volume ∼15 ml/kg; HV_T). At initiation of HV_T-ventilation, recombinant human Ang-1 was intravenously administered (1 or 4 µg per animal). Non-ventilated mice served as controls.

Results

HV_T-ventilation influenced the Ang-Tie2 system in lungs of healthy mice since Ang-1, Ang-2 and Tie2 mRNA were decreased. Treatment with Ang-1 increased Akt-phosphorylation indicating Tie2 signaling. Ang-1 treatment reduced infiltration of granulocytes and expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1 and interleukin (IL)-1β caused by HV_T-ventilation. Importantly, Ang-1 treatment did not prevent vascular leakage and impaired gas exchange in HV_T-ventilated mice despite inhibition of inflammation, vascular endothelial growth factor (VEGF) and Ang-2 expression.

Conclusions

Ang-1 treatment downregulates pulmonary inflammation, VEGF and Ang-2 expression but does not protect against vascular leakage and impaired gas exchange induced by HV_T-ventilation.     

Dimethylthiourea consumption reflects H2O2 concentrations and severity of acute lung injury

Even though dimethylthiourea (DMTU) effectively scavenges O2 metabolites in vitro, it is often unclear if scavenging of O2 metabolites is the mechanism by which DMTU decreases tissue injury in biological models. Since DMTU not only scavenges O2 metabolites but is also consumed in a dose-response manner following reaction with hydrogen peroxide (H2O2) in vitro, we wondered whether DMTU would also be consumed by O2 metabolites in biological systems and if DMTU consumption would then reflect O2 metabolite concentrations and O2 metabolite-mediated injury. Our results supported this possibility. We found that selected nonprotecting concentrations of DMTU were consumed in isolated rat lungs perfused with H2O2 and that the amounts of DMTU consumed reflected both the added amounts of H2O2 and the corresponding degrees of H2O2-induced acute edematous injury. DMTU consumption was relatively specific for reaction with H2O2 occurring in isolated lungs that were injured by H2O2 but not lungs injured by elastase, oleic acid, histamine, or a venous pressure challenge. Our results suggest that measurement of DMTU consumption may be useful for assessing the presence and toxicity of O2 metabolites and the specificity of the protective effects of DMTU in biological systems.     

Dystroglycan does not contribute significantly to kidney development or function, in health or after injury

Dystroglycan (DG or DAG1) is considered a critical link between the basement membrane and the cytoskeleton in multiple tissues. DG consists of two subunits, an extracellular α-subunit that binds laminin and other basement membrane components, and a transmembrane β-subunit. DG-null mouse embryos die during early embryogenesis because DG is required for Reichert's membrane formation. DG also forms an integral part of the dystrophin-glycoprotein complex in muscle. Although no human DG mutations have been reported, multiple forms of muscular dystrophy have been linked to DG glycosylation defects, and targeted deletion of muscle DG causes muscular dystrophy in mice. Moreover, DG is widely distributed in endothelial and epithelial cells, including those in the kidney. There has therefore been significant interest in DG's role in the kidney, especially in podocytes. Previous reports suggested that DG's disturbance in podocytes might cause glomerular filtration barrier abnormalities. To fully understand DG's contribution to nephrogenesis and kidney function, we used a conditional DG allele and a variety of Cre mice to systematically delete DG from podocytes, ureteric bud, metanephric mesenchyme, and then from the whole kidney. Surprisingly, none of these conditional deletions resulted in significant morphological or functional abnormalities in the kidney. Furthermore, DG-deficient podocytes did not show increased susceptibility to injury, and DG-deficient kidneys did not show delayed recovery. Integrins are therefore likely the primary extracellular matrix receptors in renal epithelia.     

Differential signaling through NFkappaB does not ameliorate skeletal myoblast apoptosis during differentiation

During 23A2 skeletal myoblast differentiation, roughly 30% of the population undergoes apoptosis. Further, constitutive signaling by G12V:H-Ras or Raf:CAAX abrogates this apoptosis. In this study, we demonstrate an increase in NFkappaB activity in myoblasts that have survived and are expressing muscle-specific genes. NFkappaB activity is also elevated in myoblasts expressing constitutively active G12V:H-Ras but not Raf:CAAX. Expression of a dominant negative IkappaB (IkappaB-SR) sufficient to eliminate this elevated level of NFkappaB activity, in either the 23A2 myoblasts or their G12V:H-Ras-expressing counterparts, however, does not affect survival. Furthermore, expression of a constitutively active IkappaB kinase in 23A2 myoblasts does not protect these cells from the apoptosis associated with differentiation. Since signaling by IkappaB kinase can abrogate differentiation, this result demonstrates that abrogated differentiation and abrogated apoptosis are separable phenotypes.     

Bone marrow-derived mesenchymal stem cells ameliorate hepatic ischemia reperfusion injury in a rat model

Background

Ischemia-reperfusion (I/R) injury associated with living donor liver transplantation impairs liver graft regeneration. Mesenchymal stem cells (MSCs) are potential cell therapeutic targets for liver disease. In this study, we demonstrate the impact of MSCs against hepatic I/R injury and hepatectomy.

Methodology/Principal Findings

We used a new rat model in which major hepatectomy with I/R injury was performed. Male Lewis rats were separated into two groups: an MSC group given MSCs after reperfusion as treatment, and a Control group given phosphate-buffered saline after reperfusion as placebo. The results of liver function tests, pathologic changes in the liver, and the remnant liver regeneration rate were assessed. The fate of transplanted MSCs in the luciferase-expressing rats was examined by in vivo luminescent imaging. The MSC group showed peak luciferase activity of transplanted MSCs in the remnant liver 24 h after reperfusion, after which luciferase activity gradually declined. The elevation of serum alanine transaminase levels was significantly reduced by MSC injection. Histopathological findings showed that vacuolar change was lower in the MSC group compared to the Control group. In addition, a significantly lower percentage of TUNEL-positive cells was observed in the MSC group compared with the controls. Remnant liver regeneration rate was accelerated in the MSC group.

Conclusions/Significance

These data suggest that MSC transplantation provides trophic support to the I/R-injured liver by inhibiting hepatocellular apoptosis and by stimulating regeneration.