Free radicals mediate amphetamine-induced acute pulmonary edema in isolated rat lung

Intravenous amphetamine abuse may cause serious cardiopulmonary complications via unknown mechanisms. We investigated the role of free radicals in the amphetamine-induced lung injury using isolated rat lungs. Adding amphetamine into the perfusate caused dose-dependent increases in perfusion pressure and lung weight. Amphetamine increased the filtration coefficient (K(f)) by 90 +/- 20% and 210 +/- 10% at doses of 10 microM and 50 microM, respectively, as compared to the baseline level. Pretreatment with dimethylthiourea (DMTU), an oxygen radical scavenger, abolished the pulmonary hypertension, lung weight gain, and permeability changes. We also examined the effect of amphetamine on free radical generation in polymorphonuclear leukocytes (PMN). Adding phorbol myristate acetate (PMA, 1 nM) enhanced the chemiluminescence indicating the functional viability of the isolated PMN. Amphetamine (50 microM) significantly enhanced the chemiluminescence generation of PMN by 152 +/- 26% as compared with the baseline value. Combination of amphetamine and PMA increased free radical formation by 360 +/- 85%. In summary, our results showed that amphetamine may cause acute lung injury by overproduction of free radicals. Although amphetamine can activate PMN, the source of free radicals remains to be determined.     

Simultaneous measurement of O2 radicals and pulmonary vascular reactivity in rat lung

The role of endogenous radicals in the regulation of pulmonary vascular tone was evaluated by simultaneous measurement of pulmonary artery pressure and lung radical levels during exposure of isolated rat lungs to varying inspired O2 concentrations (0-95%) and angiotensin II. Lung radical levels, measured "on-line" using luminol and lucigenin-enhanced chemiluminescence, decreased in proportion to the degree of alveolar hypoxia. Radical levels fell during hypoxia before the onset of pulmonary vasoconstriction and promptly returned to basal levels with restoration of normoxic ventilation. Mild alveolar hypoxia (10% O2), which failed to decrease chemiluminescence, did not trigger pulmonary vasoconstriction. Although chemiluminescence tended to decrease more as the hypoxic response strengthened, there was not a simple correlation between the magnitude of the change in chemiluminescence induced by hypoxia and the strength of the hypoxic pressor response. Normoxic chemiluminescence was largely inhibited by superoxide dismutase but not catalase. Superoxide dismutase also increased normoxic pulmonary vascular tone and the strength of the pressor response to hypoxia and angiotensin II. Thus the predominant activated O2 species in the lung, during normoxia, was the superoxide anion or a closely related substance. Alteration of endogenous radical levels can result in changes in vascular tone. It remains uncertain whether the decrease in lung radical production during hypoxia caused pulmonary vasoconstriction or was merely associated with hypoxic ventilation.     

Recipient T cells mediate reperfusion injury after lung transplantation in the rat

Leukocytes have been implicated in ischemia-reperfusion (IR) injury of the lung, but the individual role of T cells has not been explored. Recent evidence in mice suggests that T cells may play a role in IR injury. Using a syngeneic (Lewis to Lewis) rat lung transplant model, we observed that recipient CD4(+) T cells infiltrated lung grafts within 1 h of reperfusion and up-regulated the expression of CD25 over the ensuing 12 h. Nude rats (rnu/rnu) and heterozygous rats (rnu/+) were used to determine the role of T cells in IR injury. No significant difference in lung function was observed between nude and heterozygous recipient rats after 2 h of reperfusion. However, after 12 h of reperfusion, recipient nude rats had significantly higher oxygenation and lower peak airway pressure than recipient heterozygous rats. This was associated with significantly lower levels of IFN-gamma in transplanted lung tissue of recipient nude rats. Reconstitution of recipient nude rats with T cells from heterozygous rats restored IR injury after 12 h of reperfusion. The effect of T cells was independent of neutrophil recruitment and activation in the transplanted lung. These results demonstrate that recipient T cells are activated and mediate IR injury during lung transplantation in rats.     

Altered pulmonary epithelial permeability in canine radiation lung injury

A radioaerosol scanning technique measuring regional clearance of sodium pertechnetate (99mTcO-4) and 99mTc-labeled diethylenetriaminepentaacetate (99mTc-DTPA) was used to assess changes in canine pulmonary epithelial permeability following lung irradiation. Doses of 2,000 cGy (11 dogs), 1,000 cGy (2 dogs), and 500 cGy (2 dogs) were given in one fraction to either the entire right hemithorax (500 cGy) or the right lower lung (1,000 and 2,000 cGy). Radioaerosol scans, chest roentgenograms, and computerized tomograms (CT) were obtained before and serially after irradiation. A dose of 2,000 cGy resulted in a decrease in regional pulmonary epithelial permeability to both 99mTcO4- and 99mTc-DTPA; both showed significant decreases from the 2nd wk postirradiation onward. In comparison, CT and chest roentgenogram did not become abnormal until 7.1 +/- 2.8 (SD) and 8.2 +/- 2.6 wk, respectively. Doses of 1,000 and 500 cGy produced reversible decreases in 99mTcO4- clearance. Lung morphology showed definite changes of radiation pneumonitis after 2,000 and 1,000 cGy but not after 500 cGy at approximately 9, 17, and 12 wk postirradiation, respectively. These results suggest that dose-dependent changes in pulmonary physiology may precede obvious structural alterations in radiation lung injury.     

Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury

Serum IL-6 is increased in acute kidney injury (AKI) and inhibition of IL-6 reduces AKI-mediated lung inflammation. We hypothesized that circulating monocytes produce IL-6 and that alveolar macrophages mediate lung inflammation after AKI via chemokine (CXCL1) production. To investigate systemic and alveolar macrophages in lung injury after AKI, sham operation or 22 min of renal pedicle clamping (AKI) was performed in three experimental settings: 1) systemic macrophage depletion via diphtheria toxin (DT) injection to CD11b-DTR transgenic mice, 2) DT injection to wild-type mice, and 3) alveolar macrophage depletion via intratracheal (IT) liposome-encapsulated clodronate (LEC) administration to wild-type mice. In mice with AKI and systemic macrophage depletion (CD11b-DTR transgenic administered DT) vs. vehicle-treated AKI, blood monocytes and lung interstitial macrophages were reduced, renal function was similar, serum IL-6 was increased, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In wild-type mice with AKI administered DT vs. vehicle, serum IL-6 was increased. In mice with AKI and alveolar macrophage depletion (IT-LEC) vs. AKI with normal alveolar macrophage content, blood monocytes and lung interstitial macrophages were similar, alveolar macrophages were reduced, renal function was similar, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In conclusion, administration of DT in AKI is proinflammatory, limiting the use of the DTR-transgenic model to study systemic effects of AKI. Mice with AKI and either systemic mononuclear phagocyte depletion or alveolar macrophage depletion had reduced lung inflammation and lung CXCL1, but increased lung capillary leak; thus, mononuclear phagocytes mediate lung inflammation, but they protect against lung capillary leak after ischemic AKI. Since macrophage activation and chemokine production are key events in the development of acute lung injury (ALI), these data provide further evidence that AKI may cause ALI.     

缺血预处理对大鼠肺缺血/再灌注损伤的保护作用

目的 :观察缺血预处理 (IPC)对大鼠肺缺血 /再灌注 (I/R)损伤的保护作用 ,并初步探讨其作用机制。方法 :建立离体大鼠肺灌流模型 ,36只wistar大鼠随机分为对照组、I/R组和IPC组 ,处理完毕后分别测定平均肺动脉压(MPAP)、肺组织湿 /干重比、支气管肺泡灌洗液中肺表面活性物质磷脂及表面张力改变 ,肺组织标本送电镜检查。结果 :①电镜下观察IPC组肺损伤明显减轻。②肺组织湿 /干重比值IPC组为 4.41± 0 .2 4,显著低于I/R组 ,但仍高于缺血前 (P <0 .0 1) ;③IPC组大鼠缺血 1h后MPAP为 ( 1.88± 0 .2 9)kPa ,明显低于I/R组 (P <0 .0 1) ;④IPC组支气管肺泡灌洗液中总磷脂为 ( 2 33 .42± 14.0 5 ) μg/kg ,大聚体为 ( 10 5 .39± 6 .17) μg/kg ,与I/R组相比显著增高 ,但低于对照组 (P <0 .0 1) ,三组之间小聚体含量没有显著差异 ;⑤IPC组表面张力为 ( 36 .88± 3.49)mN/m ,显著低于I/R组 ,与对照组相比则无显著性差异 (P >0 .0 5 )。结论 :缺血预处理对大鼠肺I/R损伤有保护作用 ,保护机制可能与促进肺表面活性物质 (PS)磷脂分泌、改善PS组成 ,从而提高PS功能有关。     

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.     

Hydroxyl radicals mediate injury to endothelium-dependent relaxation in diabetic rat

The purpose of this study was to determine the radical species which mediates the toxic effects of exogenous oxygenderived free radicals on endothelial function of chronic diabetic rat aorta. Endothelium-dependent relaxation to acetylcholine was impaired in diabetic vessels. Exposure to the exogenous free radical generating system of xanthine plus xanthine oxidase selectively impaired endothelium-dependent relaxation to acetylcholine in control and diabetic aorta with relaxations essentially abolished in diabetic aorta. The loss of relaxation to acetylcholine in diabetic aorta was prevented or attenuated by pretreatment with catalase, dimethylthiourea or desferrioxamine, but not by mannitol or superoxide dismutase. These results suggest that hydroxyl radicals play an important role in the endothelial injury produced by oxygen-derived free radicals in chronic diabetic rat aorta. Furthermore, the site of the injury is likely due to intracellular generation of hydroxyl radicals.     

缺血后处理对大鼠再灌注损伤肺细胞凋亡的影响

目的：探讨缺血后处理对再灌注损伤肺细胞凋亡的影响。方法：健康雄性sD大鼠24只,随机分为对照组（C组）、缺血／再灌注组（I／n组）和缺血后处理组（IPostC组）（n=8）。对比观察各组血清中丙二醛（MDA）、超氧化物歧化酶（SOD）、髓过氧化物酶（MPO）活力及含量变化,原位缺口末端标记法（TUNEL）检测肺组织细胞凋亡情况,免疫组化及RT-PCR法检测肺组织中Bax、Bcl一2蛋白和基因的表达。结果：I／R组与c组相比,MDA含量、MPO活力明显升高,SOD活力明显下降（均P〈0．01）,肺组织原位细胞凋亡检测示I／R组凋亡指数（AI）（39．03±3．46）显著高于C组（2．88±0．34）,Bcl-2／Bax比值在蛋白和基因水平明显降低（均P〈0．01）;IPostC组与I／R组相比MDA含量显著降低（P〈0．05）,MPO活力显著降低（P〈0．01）,SOD活性升高（P〈0．01）,AI为8．03±0．88显著低于L／R组,并能明显升高Bcl-2／Bax比值（均P〈0．01）。结论：缺血后处理通过减轻脂质过氧化反应及中性粒细胞聚集,降低Bax／Bel．2比值,使肺组织细胞凋亡减少,从而有效地减轻肺缺血／再灌注损伤。     

Polymorphonuclear leukocyte cytoplasts mediate acute lung injury

Injection of phorbol 12-myristate 13-acetate (PMA) into polymorphonuclear leukocyte (PMN)-depleted, PMN cytoplast-repleted New Zealand White rabbits caused the development of acute lung injury in vivo. PMN cytoplasts are nucleus- and granule-free vesicles of cytoplasm capable of releasing toxic O2 radicals but incapable of releasing granule enzymes. PMN cytoplasts when activated by PMA reduced 66 +/- 12.7 nmol of cytochrome c compared with 2.6 +/- 0.7 nmol in their resting state and did not release a significant quantity of granule enzymes (P greater than 0.05). Injection of PMA into New Zealand White rabbits caused a significant decrease (P less than 0.05) in the number of circulating cytoplasts. Increases in lung weight-to-body weight ratios in PMA-treated rabbits (9.8 +/- 0.5 X 10(-3] compared with saline-treated rabbits (5.3 +/- 0.2 X 10(-3] were also noted. Levels of angiotensin-converting enzyme in lung lavage as well as the change in alveolar-arterial O2 ratio correlated with the numbers of cytoplasts in lung lavage (P = 0.001, r = 0.84 and P = 0.0166, r = 0.73, respectively). Albumin in lung lavage increased to 1,700 +/- 186 mg/ml in PMA-treated rabbits from 60 +/- 30 mg/ml in saline-treated rabbits. These changes were attenuated by pretreatment of rabbits with dimethylthiourea (DMTU). In vitro, cytoplasts were able to mediate increases in endothelial monolayer permeability. This was evidenced by increases in fractional transit of albumin across endothelial monolayers when treated with PMA-activated cytoplasts (0.08 +/- 0.01 to 0.28 +/- 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (K(ATP)); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 +/- 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 +/- 1.9%, 11,12-EET; and 8.4 +/- 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 +/- 2.1%, 11,12-EET; and 9.7 +/- 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 +/- 4.6%) by the K(ATP) channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 +/- 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP omega-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 +/- 1.2%, DDMS; and 22.2 +/- 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP omega-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP omega-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac K(ATP) channels.     

Nigella sativa attenuates myocardial ischemic reperfusion injury in rats

Myocardial ischemia–reperfusion (I/R) represents a clinically relevant problem associated with thrombolysis, angioplasty, and coronary bypass surgery. Radical oxygen species generated during early reperfusion are the primary activator of mitochondrial permeability transition pore (MPTP) opening which finally results in cardiomyocyte death. Nigella sativa (NS) has been shown to have antioxidant properties. The present study aimed to determine whether supplementation with NS can provide sufficient protection for the myocardium against I/R insult and any possible role on mitochondrial MPTP. Adult male Wistar rats were allocated into two groups: control group and NS-treated group receiving NS (800 mg/kg) orally for 12 weeks. Rats' isolated hearts were perfused in Langendorff preparation to determine the baseline heart beating rate, developed peak tension, time to peak tension, rate of tension development, half relaxation time, and myocardial flow rate. Ischemia was then induced by stopping the perfusion fluid for 30 min, followed by 30 min of reperfusion and recording post I/R cardiac functions. Hearts were then used for assessment of malondialdehyde (MDA) and nicotinamide adenine dinucleotide (NAD⁺), since the hydrolysis of mitochondrial NAD⁺ directly reflects MPTP opening in situ, and for histological examination. The NS-treated group showed enhanced post I/R contractile and vascular recovery, which was accompanied by elevated NAD⁺ and decreased MDA compared to the control group. Histological examination showed marked improvement of cardiac musculature compared to the control group. In conclusion, N. sativa afforded substantial recovery of post I/R cardiac functions probably via inhibition of MPTP opening.     

Spin trapping of oxygen and carbon-centered free radicals in ischemic canine myocardium

Oxygen free radical injury has been postulated to occur during myocardial ischemia. We have used Electron Spin Resonance and Spin Trapping techniques to directly demonstrate the production of carbon-centered (R.) and oxygen-centered lipid radical (RO.) in ischemic canine heart. In addition, venous effluent from the ischemic region showed that conjugated dienes (lipid peroxidation products) increased with ischemic duration. Our results suggest that the formation of the oxygen-centered and carbon-centered lipid radical species during ischemia are a consequence of oxy-radical peroxidation of myocardial membrane lipids.     

Neutrophils are primary source of O2 radicals during reperfusion after prolonged myocardial ischemia

Although many studies document oxygen radical formation during ischemia-reperfusion, few address the sources of radicals in vivo or examine radical generation in the context of prolonged ischemia. In particular, the contribution of activated neutrophils remains unclear. To investigate this issue, we developed a methodology to detect radicals without interfering with blood-borne mechanisms of radical generation. Dogs underwent aorta and coronary sinus catheterization. No chemicals were infused; instead, blood was drawn into syringes prefilled with a spin trap and analyzed by electron paramagnetic resonance spectroscopy. After 90 min of coronary artery occlusion, transcardiac concentration of oxygen radicals rose severalfold 10 min after reflow and remained significantly elevated for at least 1 h. Radicals were mostly derived from neutrophils, as shown by marked reduction after the administration of 1) neutrophil NADPH oxidase inhibitors and 2) a monoclonal antibody (R15.7) against neutrophil CD18 adhesion molecule. Reduction of radical generation by R15.7 was also associated with a significantly smaller infarct size and no-reflow areas. Thus our data demonstrate that neutrophils are a major source of oxidants in hearts reperfused in vivo after prolonged ischemia and that antineutrophil interventions can effectively prevent the increase in oxygen radical concentration during reperfusion.     

Oxygen-derived radicals: a link between reperfusion injury and inflammation

Oxygen-derived free radicals (superoxide and hydroxyl) and related species (hydrogen peroxide and hypohalous acids) have well-defined roles in the inflammatory process. Their actions include the killing of microorganisms as well as participation in cell-to-cell communication among phagocytes via the activation of a superoxide-dependent chemoattractant. The active oxygen species also have roles in postischemic injury brought about by the conversion during ischemia of the enzyme xanthine dehydrogenase (EC 1.1.1.204) to the radical-producing xanthine oxidase (EC 1.1.3.22). Although the enzymes responsible for producing superoxide in inflammation and ischemia are quite distinct, and are triggered by very different events, there are points of interplay in the two mechanisms whereby an ischemia/reperfusion-induced injury would lead to inflammation, and conversely whereby inflammation could lead to impairment of the circulation and hence to ischemic injury.     

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.