A. Verheyen

Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice

To investigate the mechanisms underlying airway hyperresponsiveness a murine model was developed with several important characteristics of human allergic asthma. Mice were intraperitoneally sensitized with ovalbumin and after 4 weeks challenge via an ovalbumin aerosol. After aerosol, lung function was evaluated with a non-invasive forced oscillation technique. The amount of mucosal exudation into the airway lumen and the presence of mast cell degranulation was determined. Tracheal responsiveness was measured at several time points after challenge. At these time points also bronchoalveolar lavage and histology were performed. Sensitization induced high antigen-specific IgE levels in serum. Inhalation of ovalbumin in sensitized mice induced an immediate but no late bronchoconstrictive response. During this immediate phase, respiratory resistance was increased (54%). Within the first hour after ovalbumin inhalation increased mucosal exudation and mast cell degranulation were observed. At 12 and 24 h after ovalbumin challenge, mice showed tracheal hyperresponsiveness (29% and 34%, respectively). However, no apparent inflammation was found in the lungs or bronchoalveolar lavage. From these results it can be concluded that hyperresponsiveness can develop via mechanisms independent of an inflammatory infiltrate. Since mast cell degranulation occurred after ovalbumin exposure, we hypothesize that mast cells are involved in the induction of airway hyperresponsiveness in this model.

Endogenous nitric oxide modulation of potassium-induced changes in guinea-pig airway tone

1 An experimental set up is used whereby the serosal (out)side or mucosal (in)side of the guinea‐pig isolated tracheal tube can be stimulated selectively with drugs and reactivity measured. 2 Potassium induces a concentration‐dependent (5 − 70 mM) monophasic contraction of tracheal tubes when added on the outside. In contrast, on the inside, potassium induces a concentration‐dependent relaxation at low concentrations (5–40 mM) which was reversed into a contraction up to approximately basal tone at higher concentrations (50–70 mM). 3 Epithelium denudation reversed the potassium‐induced relaxation into a contraction. Interestingly, in the ‘half epithelium‐denuded trachea the contractions were significantly (P<0.01) reduced by 46% compared to complete epithelium‐denuded tissues. 4 Incubation with the nitric oxide (NO) synthase inhibitor NΩ‐nitro‐L‐arginine methyl ester (l‐NAME, 120 μm) for 30 min on the inside of the tracheal tube completely prevented the relaxation. However, L‐NAME did not reverse the potassium‐induced relaxation into a contraction. This indicates that potassium does not penetrate through the epithelial layer. 5 It is concluded that depolarization of smooth muscle cells leads to a monophasic contraction and that depolarization of the epithelium leads to a relaxation of tracheal smooth muscle. The epithelial layer has an important barrier function and can release relaxing factors like NO.

Viral infection in guinea pigs induces a sustained non-specific airway hyperresponsiveness and morphological changes of the respiratory tract

In the present study an animal model is described in which a sustained non-specific airway hyperresponsiveness is induced. Guinea pigs were inoculated intratracheally with parainfluenza type 3 (PI-3) virus or control solution. Two, 4, 8, and 16 days after inoculation the tracheae, bronchi, and lung strips were isolated and mounted in organ baths. Two days after inoculation no difference between the control solution and PI-3 virus group was observed, with respect to the histamine concentration/response curve obtained from tracheae, bronchi and lung strips of the respective groups. However, histamine concentration/response curves were significantly (P less than 0.01) shifted upwards in all parts of the airways 4, 8, and 16 days after PI-3 inoculation as compared with the control solution. The excessive contraction of the trachea was not specific for histamine, since an increase in the maximal response was obtained also for the cholinergic receptor agonist, arecoline on day 4 (32%, P less than 0.05), day 8 (24%, P less than 0.05), and day 16 (28%). Morphological examination of the central airways obtained from control solution-inoculated animals revealed no signs of inflammation. However, 2, 4, and 8 days, but not 16 days, after the viral infection, epithelial damage with loss of cilia and mucus-depleted goblet cells were observed. Thus, morphological changes were not directly associated with changes in airway responsiveness. Histological examination of the peripheral airways revealed an influx of inflammatory cells, as shown by typical lesions of patchy alveolitis and bronchiolitis. Bronchiolar epithelium was variously hyperplastic and dysplastic with degenerative changes, and the lumens of the bronchioli were occluded with mucus and inflammatory cells. In conclusion, the virus-induced airway hyperresponsiveness in guinea pigs shows similarities with the human situation, in which a sustained non-specific airway hyperresponsiveness is observed after a respiratory viral infection. In addition, the hyperresponsiveness seems to be accompanied by an influx of inflammatory cells in the airways but not with other morphological changes.

Identification of macrophages in intimal thickening of rat carotid arteries by cytochemical localization of purine nucleoside phosphorylase.

Complete desquamation of the endothelium of the rat carotid artery by balloon catheter stripping resulted within 2 weeks in the formation of a large intimal thickening. After an enzyme cytochemical technique was applied to localize cytosolic purine nucleoside phosphorylase (PNP), light microscopical evaluation indicated that this intimal thickening in normocholesterolemic rats was composed of 5.8% to 11.8% (mean 8.8%) PNP-positive cells. At the electron microscopic level, all these PNP-positive cells were identified as macrophages by the absence of a basement membrane and plasmalemmal vesicles and by the occurrence of specific intracytoplasmic granules. The nearly nonreactive intimal cells were classified as modified smooth muscle cells. Additional evidence of the macrophage nature of the PNP-stained intimal cells was obtained by differential immunogold labeling of these cells with a monoclonal antibody against rat macrophages. Moreover, in hypercholesterolemic rats, only the cells stained for PNP transformed into foam cells (between 8.5% and 11.4% of all nucleated intimal cells; mean 9.6%). This study shows that PNP cytochemistry discriminates macrophages from modified smooth muscle cells in the rat carotid intimal thickening. It further suggests that the intimal thickening in normocholesterolemic rats originates not only from migration and proliferation of smooth muscle cells, but also from a considerable number of leukocyte-derived macrophages. Whether the latter cells are actively involved in the establishment of the intimal thickening as has been suggested in dietary hypercholesterolemia, remains to be verified.

Virus-induced airway hyperresponsiveness in the guinea pig can be transferred by bronchoalveolar cells

For the investigation of whether inflammatory cells were responsible for virus-induced airway hyperresponsiveness, tracheal spirals from healthy guinea pigs were incubated in organ baths with different numbers of bronchoalveolar cells obtained from guinea pigs 4 days after their inoculation with parainfluenza-3 (P-3) virus or control solution. Airway responsiveness was measured by performance of histamine concentration/response (C/R) curves on the tissues. Preparations incubated with 5 x 10(5) cells/ml obtained from guinea pigs treated with P-3 virus demonstrated a significant upward shift of the histamine C/R curve. The maximal contraction was increased by 26% as compared with the tissues incubated with the same number of cells from animals inoculated with control solution. When the number of cells was increased further to 5 x 10(6) cells/ml, no additional upward shift of the C/R curve was seen; the increase in maximal contraction was 24%. Tracheal spirals incubated with 5 x 10(4) cells/ml did not affect the histamine C/R curves. Addition of P-3 virus to the organ bath during the incubation period with the cells did not affect the histamine C/R curve either, irrespective of the inoculation solution or the number of bronchoalveolar cells used. The relative number of alveolar macrophages in bronchoalveolar lavage fluid decreased significantly from 86.3% +/- 2.6% in the control group to 71.8% +/- 3.3% in the P-3 virus group as a consequence of a significant increase in the percentage of monocytes, lymphocytes, and eosinophils. These results suggest that bronchoalveolar cells are causally involved in the virus-induced airway hyperresponsiveness.

Bovine tracheal responsiveness in vitro: role of the epithelium and nitric oxide

Airway epithelium releases inhibitory factors, such as nitric oxide (NO) and prostaglandin E2 (PGE2), which may counteract bronchoconstriction. We investigated whether epithelium-derived inhibitory substances exert a crucial influence on bovine tracheal responsiveness in vitro. Isotonic and isometric contractions in response to histamine of intact and epithelium-denuded tracheal smooth muscle strips were compared. In addition, the effects of L-arginine (L-arg), N(G)-nitro-L-arginine methyl esther (L-NAME), and N(G)-monomethyl L-arginine (L-NMMA) on histamine responsiveness were investigated. The release of NO and PGE2 from tracheal epithelium was measured. Removal of the epithelium from tracheal smooth muscle strips did not change the negative log of the concentration of histamine producing half the maximal effect (pD2) or the maximal effect (Emax). Incubation of the tissues for 25 min with L-arg or L-NAME did not influence basal tone or the contractions induced by histamine. However, incubation with L-NMMA increased the basal tone and caused a slight hyporesponsiveness to histamine. S-nitroso-N-acetyl-penicillamine (SNAP, a direct NO donor) reversed the contraction induced by histamine in a concentration-dependent manner. Stimulation of the epithelial layer by 0.1 microM histamine increased the release of NO 3-4 fold compared to basal levels; this effect was completely inhibited in the presence of L-NMMA. In addition, 1 mM histamine caused a significant increase in the release of PGE2 from the epithelial tissue. In conclusion, no functional inhibitory influence of the epithelium can be identified in bovine airways. The S-nitroso-N-acetyl-penicillamine-induced relaxation demonstrates the presence of a nitric oxide sensitive pathway in bovine airways. However, the amounts of nitric oxide and prostaglandin E2 released from bovine tracheal epithelium are probably too low to exert a significant effect on the histamine-induced contractions.

Oversensitivity to serotonin of the collateralized vascular bed in rat hindquarters: mechanisms of increased vasoconstriction

A buffered solution was perfused at a constant flow rate (2 ml/min) through both iliac arteries in rat hindquarters. Perfusion pressures were measured in normal and collateralized vascular beds of the left and right hind-leg, respectively. Bolus injections of various agonists produced concentration-dependent increases in perfusion pressure in both collateralized and normal circulatory beds. Serotonin, in particular, and noradrenaline, to a lesser extent, produced more pronounced vasoconstriction on the collateral side than on the normal side. The difference in vasoreactivity to serotonin was related to a difference in both vascular structure and sensitivity of both types of vascular bed. Vasoconstriction induced by serotonin was inhibited by 5-HT2 antagonists. Selective blockade of alpha 1,alpha 2,beta 1-beta 2 adrenoceptors and amine uptake blockade were ineffective. This study indicates that, in rat hind-legs, the collateralized vascular bed is superreactive to serotonin in comparison with the normal bed. This resetting of reactivity to serotonin is due to the specific vascular structure as well as to an increased 5-HT2 receptor-mediated sensitivity.

Effect of Ridogrel, a Combined Thromboxane A2 Synthase Inhibitor/ Prostaglandin Endoperoxide Receptor Antagonist, on the Lysis of Platelet- rich Coronary Arterial Thrombi with Recombinant Tissue-type Plasminogen Activator in a Canine Model

The effect of ridogrel, a combined thromboxane A2 synthase inhibitor/prostaglandin endoperoxide receptor antagonist, on the lysis of platelet-rich thrombi with recombinant tissue-type plasminogen activator (rt-PA) was studied in everted (inside-out) femoral arterial grafts inserted in the left anterior descending coronary arteries of heparinised dogs. Thrombotic occlusion of the everted segment graft with a platelet-rich thrombus, persisting for at least 30 min, occurred spontaneously within 4.3±3.9 min (mean±SD). These dogs were then heparinised and randomised to 1 of 4 blinded treatment groups: double placebo infusion, bolus injections of 0.5 mg/kg rt-PA, repeated at 15 min intervals until recanalisation occurred or up to 4 doses, ridogrel infusion (5 mg/kg bolus followed by continuous infusion of 5 mg/kg over 150 min), or the combination of rt-PA and ridogrel. In the control group, stable occlusion as measured with an electromagnetic flow probe was maintained throughout the observation period. rt-PA produced reperfusion in 3 of 5 dogs, associated with cyclic reocclusion and reflow in 1 dog. Ridogrel administration did not produce recanalisation in any of the animals. The combined administration of ridogrel and rt-PA produced stable reperfusion without reocclusion in all of 5 dogs (p<0.003 vs control groups), within 41±17 min. Coronary blood flow after recanalisation was significantly higher (p<0.05) in dogs given rt-PA and ridogrel (29±6 ml/min after 10 min and 30±9 ml/min after 60 min) than in dogs given rt-PA alone (10±5 ml/min after 10 min and 14±6 ml/min after 60 min). Ridogrel, alone or in combination with rt-PA, prolonged the template bleeding time from approximately 3.5 min to more than 20 min, whereas rt-PA alone did not significantly affect the bleeding time. The results indicate that ridogrel enhances and sustains recanalisation of platelet-rich arterial thrombosis with rt-PA.

Electrically induced coronary artery thrombosis in closed chest anaesthetized dogs. Evaluation of the method.

SummaryTheSalazar technique for electrical induction of coronary thrombosis, was used in 12 dogs, whereas a control procedure was followed in 6 other dogs.Electrocardiographic, hemodynamic, biochemical and hematological studies were carried out before, during and 24 hours after electrical stimulation. Chaotic rhythm appeared in all electrically stimulated dogs 24 hours after thrombus formation.Muscle necrosis was evident as indicated by the increase in SGOT and SGPT, but the observed hemodynamic changes were rather small. Light and electron microscopic studies of the coronary arteries showed important lesions in the vessel wall of the catheterized coronary arteries and an intracoronary thrombus was found in all the animals electrically stimulated as well as in two control dogs. These studies also revealed a multiple origin of coronary artery thrombus formation.This closed chest model for acute myocardial infarction is well suitable for the study of early dysrhythmias, especially because of the high incidence of dysrhythmia and the low mortality rate. The model is not suitable to study new chemical compounds with anti-thrombotic activity, because of the multiple uncontrollable origin of the coronary thrombus.ZusammenfassungBei 12 Versuchshunden wurde die Technik nachSalazar zur Erzeugung von elektrisch induzierten Koronarthrombosen angewandt. Als Kontrolle dienten weitere 6 Hunde.Elektrokardiographische, hämodynamische, biochemische und hämatologische Untersuchungen wurden vor, während und 24 Stunden nach elektrischer Reizung durchgeführt. Bei allen Versuchshunden (mit elektrischer Reizung) traten innerhalb von 2 Stunden nach Thrombusbildung Rhythmusstörungen auf.Aufgrund des Anstiegs von SGOT und SGPT wurden Myokardnekrosen nachgewiesen, dagegen waren die beobachteten hämodynamischen Veränderungen sehr gering. Licht- und elektronenmikroskopische Untersuchungen der Koronararterien zeigten schwere Läsionen der Gefäßwände nach Katheterisierung der Koronararterien. Ein intrakoronarer Thrombus wurde bei allen Tieren nach Anwendung elektrischer Ströme gefunden, ebenso jedoch auch bei 2 Kontrollhunden. Diese Untersuchungen zeigen, daß für die Bildung von Thrombosen der Koronararterien unterschiedliche Ursachen verantwortlich zu machen sind.Dieses Myokardinfarkt-Modell am nicht eröffneten Thorax ist zur Untersuchung frühzeitiger Rhythmusstörungen geeignet, besonders wegen des häufigen Auftretens der Dysrhythmien bei niedriger Mortalität. Das Modell erscheint jedoch nicht dafür geeignet, neue Arzneimittel mit antithrombotischer Wirkung zu testen, weil die Ursache der Thrombusbildung vielfältig und nicht kontrollierbar ist.

Differential Vasoreactivity to the Thromboxane A2 Mimic U-46619 of Collateral and Normal Peripheral Blood Vessels in in situ Perfused Rat Hindquarters

In situ perfusion of rat hindquarters was performed through both iliac arteries. Perfusion pressures were measured concomitantly in the normal vascular bed and in the ligation-induced collateral bed of the left and right hindleg, respectively. An increased vaso-constrictive response of the collateral versus normal blood vessels was found after bolus injections of increasing concentrations of the thromboxane A2 (TXA2) mimic U-46619. This increased reactivity was related to a difference in vascular structure and in receptor-mediated sensitivity. The vasoconstrictive effect of U-46619 was dose-dependently inhibited by the combined TXA2 synthetase inhibitor-TXA2/prostaglandin endoperoxide (PGH2) receptor antagonist ridogrel and the single TXA2/PGH2 receptor antagonist sulotroban. Selective blockade of alpha 1-, alpha 2-, beta 1-beta 2- and S2-receptors, cyclooxygenase inhibition, TXA2 synthesis inhibition, amine depletion and amine uptake blockade had no influence on the vasoconstrictive action of U-46619. Ca2+ entry blockade by flunarizine and nifedipine significantly reduced the vasoconstriction. This study shows that (1) rat peripheral collaterals in relation to normal arterial blood vessels are significantly more reactive to TXA2; (2) the vasoconstrictive effect of U-46619 is mediated predominantly by TXA2/PGH2 receptors; (3) the activation of these receptors elicits indirectly transmembrane Ca2+ entry to trigger vasoconstriction.