Genetic analysis of ozone-induced acute lung injury in sensitive and resistant strains of mice

Sprague-Dawley rats sedated with intraperitoneal injection of diazepam (7.5 mg/kg) were placed in a plethysmograph to measure the changes in spontaneous respiration. Inhalation of methacholine (MCh) or acetylcholine (ACh) aerosol did not alter the volume of breathing, but increased respiratory frequency (RF) to the same extent in a concentration-dependent manner. On the other hand, the tachypnea effect of MCh lasted 11 min, and that of ACh only 3 min. Urethane anesthesia inhibited spontaneous respiration and the response to MCh. Atropine, salbutamol and aminophylline inhibited MCh-induced tachypnea. In sensitized rats, the response to MCh was potentiated 6 h after inhalation of ovalbumin aerosol. The results indicate that sedation with diazepam and inhalation of MCh aerosol used in this report are suitable for measuring airway responsiveness in terms of degree of increase of respiratory frequency.     

Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury

The role of nitric oxide (NO) in lung injury remains unclear. Both beneficial and detrimental roles have been proposed. In this study, we used mutant mice lacking the inducible nitric oxide synthase (iNOS) to assess the role of this isoform in sepsis-associated lung injury. Wild-type and iNOS knockout mice were injected with either saline or Escherichia coli endotoxin (LPS) 25 mg/kg and killed 6, 12, and 24 h later. Lung injury was evaluated by measuring lactate dehydrogenase activity in the bronchoalveolar lavage, pulmonary wet/dry ratio, and immunostaining for nitrotyrosine formation. In the wild-type mice, LPS injection elicited more than a 3-fold rise in lactate dehydrogenase activity, a significant rise in lung wet/dry ratio and extensive nitrotyrosine staining in large airway and alveolar epithelium, macrophages, and pulmonary vascular cells. This was accompanied by induction of iNOS protein and increased lung nitric oxide synthase activity. By comparison, LPS injection in iNOS knockout mice elicited no iNOS induction and no significant changes in lung NOS activity, lactate dehydrogenase activity, lung wet/dry ratio, or pulmonary nitrotyrosine staining. These results indicate that mice deficient in iNOS gene are more resistant to LPS-induced acute lung injury than are wild-type mice.     

Effects of triiodothyronine (T3) supplementation upon ozone-induced lung injury

Ozone exposure results in an acute decrease in the serum levels of thyroid hormones; the physiologic sequelae of this are unclear. Whereas thyroid hormone supplementation appears to benefit pulmonary function in septic, oxyradical models of injury, thyroid hormone increases ozone toxicity. We demonstrated an increase in metabolic rate and pulmonary injury in lungs from ozone exposed, T3 treated animals. This was evidenced by an increase in pulmonary weight gain, vascular perfusion pressure, and decrease in compliance in the supplemented animals. However, an increase in alkane generation, as an index of lipid peroxidation, was not seen in the ozone exposed, hormonally treated animals. This suggests that although thyroid hormone supplementation increases metabolic rate and ozone toxicity, an increased rate of lipid peroxidation plays a minimal role.     

Effects of pretreatment with SDZ MRL 953, a novel immunostimulatory lipid A analog, on endotoxin-induced acute lung injury in guinea pigs

SDZ MRL 953 (SDZ), a novel immunostimulatory lipid A analog, has been reported to have immunopharmacological activities similar to those of lipopolysaccharide (LPS) but to have little of the toxicity of LPS. We investigated the effects of pretreatment with SDZ on Escherichia coli endotoxin-induced acute lung injury in guinea pigs. Four experimental groups consisted of saline control (n = 16), SDZ (-12 h) plus LPS (2 mg/kg of SDZ per kg of body weight injected intravenously 12 h before intravenous injection of 2 mg of LPS per kg; n = 15), SDZ (-10 min) plus LPS (SDZ injected 10 min before LPS injection; n = 10), and LPS alone (n = 16). The animals were sacrificed, and lung tissue was sampled 4 h after LPS or saline infusion. Lung injury was assessed by measuring the wet weight-to-dry weight ratio and the level of 125I-labeled albumin accumulation in bronchoalveolar lavage fluid relative to that in plasma. In the SDZ (-12 h) plus LPS group, these two parameters of acute lung injury were decreased compared with those in the LPS alone group. However, they were not decreased in the SDZ (-10 min) plus LPS group. We conclude that SDZ attenuates endotoxin-induced acute lung injury when it is administered 12 h before LPS injection. The attenuating effects of SDZ are speculated to be due to down regulation of the response to endotoxin rather than to receptor blocking.     

A possible involvement of oxidative lung injury in endotoxin-induced bronchial hyperresponsiveness to substance P in guinea pigs

Fragile-X syndrome and myotonic dystrophy are caused by triplet repeat expansions embedded in CpG islands in the transcribed non-coding regions of the FMR1 and the DMPK genes, respectively. Although initial reports emphasized differences in the mechanisms by which the expanded triplet repeats caused these diseases, results published in the past year highlight remarkable parallels in the likely molecular etiologies. At both loci, expansion is associated with altered chromatin, aberrant methylation, and suppressed expression of the adjacent FMR1 and DMAHP genes, implicating epigenetic mediation of these genetic diseases.     

Effect of burn injury on glucose turnover in guinea pigs

The Reflomat System for rapid estimation of plasma or blood glucose concentration has been evaluated. The System gave a linear response throughout its analytical range and the recovery of glucose added to glucose-free plasma was 97-105%. Addition of sodium fluoride to plasma produced a 7-15% reduction in the estimated glucose concentration. Plasma glucose concentration estimated with the Reflomat agreed closely with results of a glucose oxidase and a hexokinase based method, and blood glucose concentration measured with the Reflomat agreed well with results of a glucose oxidase method.     

Role of vagus nerves in resiniferatoxin-induced bronchoconstriction of guinea pigs

To study the role of vagal afferent C-fibers in resiniferatoxin (RTX)-induced bronchoconstriction in vivo, 30 guinea pigs weighing 347 +/- 28 g were evenly and randomly divided into five groups: Group 1, control; 2, chronic vagotomy; 3, local capsaicin (acute); 4, local capsaicin (chronic); and 5, systemic capsaicin. Each animal was anesthetized with pentobarbital sodium, cannulated with a tracheal cannula and venous catheter, paralyzed with gallamine triethiodide, and artificially ventilated. All animals were pretreated with atropine and phenoxybenzamine. Immediately after RTX was intravenously injected, each animal in the control group exhibited profound decreases in maximal expiratory flow, dynamic respiratory compliance, and total lung capacity, as well as an increase in functional residual capacity, indicating severe airway constriction. Animals in Groups 2-4 exhibited partial abolishment, while those in Group 5 showed complete abolishment of the RTX-induced bronchoconstriction. In 12 additional animals (6 animals each in control and chronic vagotomy groups), chronic vagotomy caused also suppressive effects on capsaicin-induced airway constriction. At one min, our data demonstrate that 36-51% of noncholinergic bronchoconstriction is due to the vagal component while the remaining constriction is due to the nonvagal component. Thus, the nonvagal component plays a significant role in this type of tachykinin-mediated airway constriction.     

Role of kinins in the vascular extravasation evoked by antigen and mediated by tachykinins in guinea pig trachea

Tachykinins released from sensory nerves mediate, at least in part, the plasma extravasation induced by allergen challenge to the airways of sensitized guinea pigs. We investigated the role of kinins in this activation of sensory nerves. We found that the increase in Evans blue dye extravasation evoked by aerosol of bradykinin (100 microM, 2 min) in the presence of phosphoramidon (2.5 mg/kg, i.v.) was abolished completely by the selective B2 bradykinin antagonist, HOE 140 (0.1 mumol/kg, i.v.), and was inhibited (60%) by the selective NK1 tachykinin receptor antagonist, CP-96,345 (2 mumol/kg, i.v.). Plasma extravasation evoked by aerosolized substance P (10 microM/kg, 2 min) in presence of phosphoramidon was abolished by CP-96,345, but was not affected by HOE 140. The extravasation of the Evans blue dye evoked by OVA (5%, 2 min) in sensitized guinea pigs was reduced by HOE 140 (45%) when the animals were perfused after 5 min and by 39% when perfusion was performed at 10 min. In the presence of phosphoramidon, the response to OVA at 10 min was reduced by 57% by HOE 140 and by 72% by CP-96,345. The combination of CP-96,345 and HOE 140 did not further increase the inhibition obtained with CP-96,345 alone. The results provide evidence that the activation of sensory nerves that contribute to Ag-evoked plasma extravasation is due to kinin release. The contribution of this cascade of events may be exaggerated in pathophysiologic conditions in which neutral endopeptidase is down-regulated.     

Effect of clenbuterol on sulfur dioxide-induced acute bronchitis in guinea pigs

When guinea pigs were exposed to sulfur dioxide (SO2) gas (800 ppm, 2 h), they showed hyperresponsiveness to intravenously administered serotonin (5-hydroxytryptamine (5-HT)). This hyperresponsiveness continued for over 24 h after the exposure to the gas. The degeneration, desquamation of epithelium, and edema of the lamina propria of the trachea and bronchi were observed in animals after a 2-h exposure of SO2 histopathologically. These changes seemed to be the early phase of acute bronchitis. Then, we examined the effect of clenbuterol, a selective beta-2 adrenoceptor agonist, on the SO2-induced bronchial hyperresponsiveness in these animals. Orally administered clenbuterol (1-10 micrograms/kg) suppressed the hyperresponsiveness to 5-HT in a dose-dependent manner. These results suggest that clenbuterol might inhibit the hyperresponsiveness that accompanies acute bronchitis and that this agent may be useful for remission of broncho-spasm.     

Role of neutrophil elastase in allergen-induced airway microvascular leakage in sensitized guinea pigs

To determine the role of neutrophil elastase in allergen-induced airway microvascular leakage, we assessed vascular permeability of guinea pig trachea by measuring the extravasation of Evans blue dye in the circulating blood. Inhalation of ovalbumin (OA) to guinea pigs sensitized with OA caused Evans blue extravasation, indicating an increased microvascular permeability. Pretreatment with ONO-5046 a specific inhibitor of neutrophil elastase, inhibited OA-induced vascular leakage in a dose-dependent manner. Tracheal instillation of human neutrophil elastase likewise increased microvascular permeability, and this effect was almost completely abolished by ONO-5046. Challenge with OA increased the number of neutrophils and neutrophil elastase activity in the bronchoalveolar lavage fluid, and these effects were inhibited by ONO-5046. These results suggest that neutrophil accumulation into the airway and the subsequent release of neutrophil elastase may play a role in the airway microvascular leakage produced by antigen challenge.     

Myopathy in guinea pigs

Myopathy resembling nutritional muscular dystrophy occurred in a colony of 150 guinea pigs. Of 54 animals affected, 27 died. Major clinical signs were depression, conjunctivitis, and reluctance to move. Lesions were widespread throughout skeletal and cardiac musculature. Clinical signs and deaths ceased when the diet was changed to a different commercial ration. A single intramuscular injection of sodium selenite and alphatocopherol brought prompt remission of clinical signs in one group of 20 so treated.     

Role of tachykinins in distilled water-induced bronchoconstriction in guinea-pigs

BACKGROUND: An inhalation of ultrasonically nebulized distilled water (UNDW) induces bronchoconstriction only in asthmatics, but the mechanism underlying the response is not fully understood. We recently showed that bronchoconstriction occurs immediately after UNDW is inhaled 20min after an aerosolized antigen challenge in passively sensitized guinea-pigs. OBJECTIVE: This study was conducted to examine the role of tachykinins in this response. METHODS: Passively sensitized animals were anaesthetized and artificially ventilated, and changes in pressure at the airway opening (Pao) were measured as an overall index of airway narrowing. A tachykinin NK1 and NK2 dual receptor antagonist, FK224, and a tachykinin NK1 selective antagonist, FK888, were intravenously administered 15 min after the antigen challenge. The effects of capsaicin desensitization and a neutral endopeptidase inhibitor, phosphoramidon, were also examined. RESULTS: FK224 and FK888 significantly (P < 0.05 and P < 0.05, respectively) reduced the time course curve of the increase in Pao caused by UNDW inhalation in a dose-dependent manner. The percentage increase in Pao from the preantigen challenge value at 1 min after the UNDW inhalation was 267.4+/-17.1, 358.0+/-33.7 and 412.4+/-27.6% with 10 mg/kg of FK224, 1.0 mg/kg of FK224 and vehicle, respectively, (P<0.01 between 10 mg/kg of FK224 and vehicle) and the value was 254.4+/-48.5% with 10 mg/kg of FK888, 327.1+/-57.6% with 1.0 mg/kg of FK888 and 418.5+/-39.0% with vehicle, respectively (P < 0.05 between 10 mg/kg of FK888 and vehicle). The capsaicin desensitization, but not phosphoramidon, significantly reduced the UNDW-induced increase in Pao. CONCLUSION: These results suggest that tachykinins, at least substance P, are involved in a part of the UNDW-induced bronchoconstriction in our guinea-pig model.     

Release of lactate by the lung in acute lung injury

The pathogenesis of hyperlactatemia during sepsis is poorly understood. We have previously described an increase in lactate concentration across the lung in the dog during early endotoxemia. Accordingly, we sought to determine if the lung releases lactate in humans and what relation this has with lung injury. METHODS: We measured lactate concentrations across the lung and lung injury scores (LIS) in two groups of patients. Group 1 consisted of nine patients with acute lung injury (LIS > or = 2.0) and elevated lactate concentrations (> 2.0 mmol/L). Group 2 contained 12 patients with no acute lung injury (LIS scores < or = 1.5), with or without increased lactate concentrations. Simultaneous measurements of plasma lactate and blood gases were obtained from indwelling arterial and pulmonary artery catheters. Measurements of cardiac output were also obtained. Lactate measurements were done using a lactate analyzer (YSI; Yellow Springs, Ohio). RESULTS: For each patient with acute lung injury and hyperlactatemia, an arterial-venous lactate gradient existed demonstrating release of lactate by the lung. This gradient persisted after correction for changes in hemoconcentration across the lung. The lactate gradient across the lung was 0.4 +/- 0.2 mmol/L for group 1 vs 0.05 +/- 0.1 mmol/L for group 2 (p = 0.001). This corresponded to a mean pulmonary lactate flux of 231.3 +/- 211.3 vs 5.0 +/- 37.2 mmol/h (p = 0.001). The lactate flux and the arterial-venous lactate difference correlated with LIS both for the entire sample and for the subgroup with hyperlactatemia (r = 0.69, p < 0.01). Pulmonary lactate flux was not related to arterial lactate levels (r = 0.25). CONCLUSION: In patients with acute lung injury and hyperlactatemia, the lung is a major source of lactate and lactate flux correlates with LIS. This lactate flux could explain some of the hyperlactatemia seen in sepsis.     

Participation of tachykinins in experimental models of lung damage

In the present investigation a possible involvement of tachykinins during sulfur dioxide-(SO2) and metabisulfite-(MBS) induced bronchoconstriction, and paraquat (PQ)-induced mortality was studied. SO2 (250ppm) inhalation and MBS (3mM) perfusion induced a marked decrease of compliance and conductance in the isolated and perfused lung. SO2-induced bronchoconstriction was associated with release of Calcitonin Gene Related Peptide, suggesting activation of capsaicin sensitive sensory nerves. Pretreatment of animals with capsaicin, in order to deplete the tachykinin content of sensory nerves, significantly reduced SO2- and MBS-induced bronchoconstriction. PQ (25mg/Kg) treatment induced high mortality (75%) after 3 weeks. Pretreatment with capsaicin significantly protected versus PQ induced mortality (25%). The results suggest that tachykinin content in the respiratory airways participate to SO2- and MBS-induced bronchoconstriction and PQ mortality.     

Chlorine gas induced acute lung injury in isolated rabbit lung

This study was designed to investigate the pathogenesis of chlorine gas (Cl2) induced acute lung injury and oedema. Isolated blood-perfused rabbit lungs were ventilated either with air (n=7) or air plus 500 parts per million (ppm) of Cl2 (n=7) for 10 min. Capillary pressure, measured by analysing the pressure/time transients of pulmonary arterial, venous and double (both arterial and venous) occlusions, was unchanged in both groups. In Cl2-exposed lungs, the fluid filtration rate increased from -0.228+/-0.25 to 1.823+/-1.23 mL min(-1) x 100 g(-1) (p<0.001) and the filtration coefficient increased from 0.091+/-0.01 to 0.259+/-0.07 mL x min(-1) x cmH2O(-1) x 100 g(-1) (p<0.001). No changes were observed in the control lungs. The extravascular lung water/blood-free dry weight ratio was 8.6+/-1.6 in the Cl2 group and 4.0+/-0.5 in the control group (p<0.001), confirming that the increase in lung weight was related to accumulation of extravascular fluid. Although the alveolar flooding by oedema is explained, in part, by the Cl2-induced epithelial injury, our results suggest that Cl2 exposure induces acute lung injury and oedema due to an increased microvascular permeability.     

Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury

Substance P, acting via the neurokinin 1 receptor (NK1R), plays an important role in mediating a variety of inflammatory processes. However, its role in acute pancreatitis has not been previously described. We have found that, in normal mice, substance P levels in the pancreas and pancreatic acinar cell expression of NK1R are both increased during secretagogue-induced experimental pancreatitis. To evaluate the role of substance P, pancreatitis was induced in mice that genetically lack NK1R by administration of 12 hourly injections of a supramaximally stimulating dose of the secretagogue caerulein. During pancreatitis, the magnitude of hyperamylasemia, hyperlipasemia, neutrophil sequestration in the pancreas, and pancreatic acinar cell necrosis were significantly reduced in NK1R-/- mice when compared with wild-type NK1R+/+ animals. Similarly, pancreatitis-associated lung injury, as characterized by intrapulmonary sequestration of neutrophils and increased pulmonary microvascular permeability, was reduced in NK1R-/- animals. These effects of NK1R deletion indicate that substance P, acting via NK1R, plays an important proinflammatory role in regulating the severity of acute pancreatitis and pancreatitis-associated lung injury.     

Mycobacterial infection in guinea pigs

We described published reports of the chaos which exists in research concerning laboratory animal models for assay of tuberculosis (TB) vaccines and proposed a "rational animal model" as a solution to the problem. This animal model, an aerosol challenge model in guinea pigs, was recently applied to the problem of differences in growth characteristics of sputum isolates of low and high virulence. The same model was used to investigate the protective effect of high dose BCG given aerogenically. Based on studies in the guinea pig model of experimental airborne TB, and a review of the literature on pathogenesis of human TB, we described an "integrated model" for the pathogenesis of TB, a model which includes a role for both the endogenous reactivation and the exogenous reinfection pathways. Our hypothesis is that tubercle bacilli must be able to gain access to the "vulnerable region" in the lung apex in order to survive the effects of the CMI response. In endogenous reactivation TB (virulent tubercle bacilli), this access occurs via the bloodstream. Whereas in exogenous reinfection TB, access to the vulnerable region occurs via multiple exposures via the respiratory tract. Central to our perspective is the acceptance of the evidence that during first infection with virulent organisms, tubercle bacilli enter the bloodstream via the efferent lymphatics. We believe the hypotheses we have proposed have the potential to lead to a further increase in our knowledge of these mechanisms and are a prerequisite to studies aimed at the development of new vaccines.