Kazunori Murakami

Role of neutrophils in spinal cord injury in the rat

Activated neutrophils are thought to be involved in tissue injury through the release of various inflammatory mediators. To understand the role of neutrophils in spinal cord injury, the effects of nitrogen mustard-induced leukocyte depletion and the administration of an anti-P-selectin monoclonal antibody on motor disturbances observed following spinal cord compression were examined in rats. Spinal cord injury was induced by applying a 20-g weight for 20 min at the level of the 12th thoracic vertebra, resulting in motor disturbances of the hindlimbs 24 h postcompression. Motor disturbances, evaluated using Tarlovs index, an inclined-plane test and climbing ability, were markedly attenuated in rats with nitrogen mustard-induced leukocytopenia. Administration of the anti-P-selectin monoclonal antibody, by which adhesion of activated neutrophils to endothelial cells may be inhibited, also attenuated motor disturbances. Histological examination revealed that intramedullary hemorrhages observed 24 h after compression at the 12th thoracic vertebra of the spinal cord were significantly attenuated in leukocytopenic animals and those which received the anti-P-selectin monoclonal antibody. The accumulation of neutrophils at the site of compression, as evaluated by measuring the tissue myeloperoxidase activity, significantly increased with time following the compression, peaking at 3 h postcompression. Spinal cord myeloperoxidase activity did not increase in sham-operated animals. Leukocyte depletion and administration of the anti-P-selectin monoclonal antibody both reduced the accumulation of neutrophils in the damaged spinal cord segment 3 h postcompression. These observations strongly suggest that activated neutrophils play an important role in compression-induced thoracic spinal cord injury and that a P-selectin-mediated interaction between activated neutrophils and endothelial cells may be a critical step in endothelial cell injury leading to spinal cord injury.

Heparin nebulization attenuates acute lung injury in sepsis following smoke inhalation in sheep.

Pseudomonas pneumonia is a common complication of smoke inhalation injury. Airway casts formed from clotted mucous occur frequently in this condition. A recent report shows that intravenous heparin improves oxygenation and reduces lung damage in a sheep model of smoke inhalation. We hypothesized that nebulized heparin could be an effective means of reducing cast formation. Female sheep (n = 19) were surgically prepared for a study of acute lung injury (ALI). After a tracheotomy, 48 breaths of cotton smoke (<40°C) were inflated into the airway. Afterwards, live Pseudomonas aeruginosa (5 × 1011 CFU) was instilled into the lung. All sheep were mechanically ventilated with 100% O2 and were divided into four groups: a heparin-nebulized group (n = 5; animals received aerosolized heparin [10,000 I.U.] 1 h after the bacterial instillation and subsequently every 4 h thereafter), an intravenous heparin group (n = 5,300U/kg/23 h, infusion was started 1 h after the injury), a saline-nebulization group (n = 5; animals received inhaled nebulized saline), and a sham injury group (n = 4, treated in the same fashion, but no injury). The animals were sacrificed after 24 h of mechanical ventilation, and lung samples were harvested. Sheep exposed to lung injury presented with typical hyperdynamic cardiovascular changes and a corresponding drop in PaO2. These changes were significantly attenuated in the heparin groups. Histological changes consisting of cellular infiltrates, lung edema, congestion, and cast formation were reduced by heparin. These data suggest that nebulized inhaled heparin is a beneficial therapy for sepsis-induced ALI.

Effects of various doses of antithrombin III on endotoxin-induced endothelial cell injury and coagulation abnormalities in rats

We previously demonstrated that antithrombin III reduced the injury to endothelial cells caused by activated leukocytes in rats administered endotoxin. This occurred via the increase of the endothelial release of prostaglandin I2, which is a potent inhibitor of leukocyte activation. We evaluated the dose of antithrombin III required to prevent such endothelial cell injury in rats administered endotoxin, by comparing the effects of various antithrombin II doses on the pulmonary vascular injury. The intravenous administration of endotoxin, 5 mg/kg, produced a transient accumulation of leukocytes in the lung, followed by pulmonary vascular injury, as indicated by an increase in the pulmonary vascular permeability, and coagulation abnormalities. The dose of 250 U/kg significantly inhibited all such effects of endotoxin. While lower doses of antithrombin III (50 and 100 U/kg) significantly inhibited such coagulation abnormalities, they failed to prevent either the pulmonary accumulation of leukocytes or the subsequent pulmonary vascular injury. Rats administered endotoxin exhibited an accumulation of neutrophils and edematous changes in the pulmonary interstitial space. Although such changes were reduced after 250 U/kg of antithrombin III, they were unaffected by lower doses of 50 and 100 U/kg. Plasma levels of 6-keto-PGF1alpha were markedly increased in rats 90 min after the administration of endotoxin, and were significantly decreased in the endotoxin-treated rats administered the lower doses of antithrombin III (50 and 100 U/kg), but not altered in those endotoxin-treated rats receiving 250 U/kg of antithrombin III. These findings suggest that a higher antithrombin III dose is necessary to prevent endothelial cell injury than is required to inhibit coagulation abnormalities in an animal model of sepsis. These observations support the notion that antithrombin III may prevent endotoxin-induced endothelial cell injury by promoting endothelial release of prostaglandin I2 and thus inhibiting leukocyte activation.

Role of neutrophil elastase in compression-induced spinal cord injury in rats.

We have previously demonstrated the importance of activated neutrophils in compression-induced spinal cord injury (SCI) in rats. In the present study, we investigate the action of neutrophil elastase in posttraumatic SCI, using two neutrophil elastase inhibitors (Eglin C and L658,758). SCI was induced by applying a 20-g weight to the spinal cord for 20 min at the level of T12, resulting in hindlimbs motor disturbances, which, when evaluated using a inclined-plane test, were significantly attenuated by Eglin C or L658,758. Histologic examination revealed that intramedullary hemorrhages observed 24 h after trauma were markedly attenuated in these agents. These inhibitors also significantly decreased neutrophil accumulation as shown by myeloperoxidase activity in the damaged spinal cord segment. Induction of leukocytopenia had the same effects as Eglin C or L658,758. These findings implicated neutrophil elastase in SCI. The enzyme may induce vascular damage leading to spinal cord ischemia.

Recombinant human activated protein C improves pulmonary function in ovine acute lung injury resulting from smoke inhalation and sepsis

Objective:To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge. Design:Prospective, randomized, controlled, experimental animal study with repeated measurements. Setting:Investigational intensive care unit at a university hospital. Subjects:Eighteen sheep (37.2 ± 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2–5 × 1011 colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 &mgr;g/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringers lactate solution to maintain constant pulmonary artery occlusion pressure. Measurements and Main Results:In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in Pao2/Fio2 ratio (control group values were 521 ± 22 at baseline [BL], 72 ± 5 at 12 hrs, and 74 ± 7 at 24 hrs, vs. rhAPC group values of 541 ± 12 at BL, 151 ± 29 at 12 hours [p < .05 vs. control], and 118 ± 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 ± 0.02, and at 24 hrs, 0.65 ± 0.08; rhAPC group at BL, 0.24 ± 0.04, and at 24 hrs, 0.45 ± 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 ± 1, and at 24 hrs, 36 ± 4; rhAPC group at BL, 21 ± 1, and at 24 hrs, 28 ± 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 ± 2; control, 17 ± 1; rhAPC, 12 ± 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals). Conclusions:Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

Aerosolized tissue plasminogen inhibitor improves pulmonary function in sheep with burn and smoke inhalation

Acute respiratory distress syndrome is a major complication in patients with thermal injury. The obstruction of the airway by cast material, composed in part of fibrin, contributes to deterioration of pulmonary gas exchange. We tested the effect of aerosol administration of tissue plasminogen activator, which lyses fibrin clots, on acute lung injury in sheep that had undergone combined burn/smoke inhalation injury. Anesthetized sheep were given a 40% total body surface, third degree burn and were insufflated with cotton smoke. Tissue plasminogen activator (TPA) was nebulized every 4 h at 1 or 2 mg for each nebulization, beginning 4 h after injury. Injured but untreated control sheep developed multiple symptoms of acute respiratory distress syndrome: decreased pulmonary gas exchange, increased pulmonary edema, and extensive airway obstruction. These control animals also showed increased pulmonary transvascular fluid flux and increased airway pressures. These variables were all stable in sham animals. Nebulization of saline or 1 mg of TPA only slightly improved measures of pulmonary function. Treatment of injured sheep with 2 mg of TPA attenuated all the pulmonary abnormalities noted above. The results provide evidence that clearance of airway obstructive cast material is crucial in managing acute respiratory distress syndrome resulting from combined burn and smoke inhalation injury.

Endotoxin-induced pulmonary vascular injury is mainly mediated by activated neutrophils in rats

Acute respiratory failure is a common complication in patients with disseminated intravascular coagulation associated with sepsis. To elucidate the role of coagulation abnormalities in acute lung injury in sepsis, we investigated the effect of anticoagulants on the pulmonary vascular injury in rat induced by lipopolysaccharide (LPS). When administered intravenously, LPS (5 mg/kg body weight) significantly increased the accumulation of 111indium-labeled neutrophils in lung 30 min after administration. Subsequently, the pulmonary vascular permeability and the serum level of fibrin and fibrinogen degradation products (E) [FDP (E)] increased and remained elevated for several hours. Neither heparin alone, heparin plus antithrombin III, or dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa, a selective inhibitor of thrombin generation, prevented LPS-induced vascular injury 6 hours after LPS administration, whereas these substances significantly inhibited the increase in serum FDP (E) at that time. LPS-induced pulmonary vascular injury was significantly attenuated in rats with methotrexate-induced leukocytopenia or treated with ONO-5046, a potent granulocyte elastase inhibitor, although ONO-5046 did not inhibit the LPS-induced increase in serum FDP (E). Thus, activated leukocytes play a more important role than coagulation abnormalities in the pathogenesis of LPS-induced pulmonary vascular injury in an experimental rat model of endotoxemia.

Inhibition of poly (ADP-ribose) polymerase attenuates acute lung injury in an ovine model of sepsis.

It is known that in various pathophysiological conditions, reactive oxidants cause DNA strand breakage and subsequent activation of the nuclear enzyme poly(ADP ribose) polymerase (PARP). Activation of PARP results in cellular dysfunction. We hypothesized that pharmacological inhibition of PARP reduces the damage in the ovine model of acute lung injury (ALI). After smoke inhalation, Pseudomonas aeruginosa (5 × 109 cfu/kg) was instilled into both lungs. All of the animals were mechanically ventilated with 100% O2. The infusion of the PARP inhibitor (INO-1001, n = 6) began 1 h after the injury and thereafter through 24 h (3 mg bolus + 0.3 mg/kg/h, i.v.). Control animals (n = 6) were treated with saline. Sham injury animals (n = 8) received sham smoke and were mechanically ventilated in the same fashion. One-half of those sham animals (n = 4) were given the same dose of INO-1001. PaO2/FiO2 ratio at 24 h in saline and in the INO-1001-treated groups were 95 ± 22 and 181 ± 22, respectively (P < 0.05). Peak airway pressure at 24 h in the saline- and INO-1001-treated groups was 32.6 ± 3.0 and 24.4 ± 2.2, respectively (P < 0.05). Pulmonary shunt fraction was also significantly attenuated. INO-1001 treatment reduced pulmonary histological injury and attenuated poly (ADP-ribose) accumulation in the lung. In conclusion, inhibition of PARP improved the ALI after smoke inhalation and pneumonia. The results suggest that the activation of PARP plays a role in the pathophysiology of ALI in sheep.

Rebamipide Attenuates Indomethacin-Induced Gastric Mucosal Lesion Formation by Inhibiting Activation of Leukocytes in Rats

Granulocyte elastase released from activatedleukocytes plays an important role in leukocyteinfiltration. Since activated leukocytes have been shownto be involved in the pathogenesis of gastric mucosal lesion formation induced by nonsteroidalantiinflammatory drugs, inhibition of granulocyteelastase release from activated leukocytes may be usefulin the prevention of these lesions. Rebamipide, a novel antiulcer agent, inhibited granulocyte elastaserelease from activated neutrophils in vitro. Rebamipideand ONO-5046, a granulocyte elastase inhibitor, markedlyinhibited gastric mucosal lesion formation in rats. Gastric myeloperoxidase activity wassignificantly increased 3 hr after indomethacinadministration. This increase was significantlyinhibited by rebamipide and ONO-5046. Cimetidine did notinhibit granulocyte elastase release from activatedneutrophils. Although cimetidine markedly prevented theindomethacin-induced gastric mucosal lesion formation,it did not reduce the gastric myeloperoxidase activity. Therefore, unlike cimetidine, rebamipide mayprevent indomethacin-induced gastric mucosal lesionformation by inhibiting neutrophil activation.

Gabexate mesilate, a synthetic protease inhibitor, attenuates endotoxin-induced pulmonary vascular injury by inhibiting tumor necrosis factor production by monocytes.

OBJECTIVE In order to determine whether gabexate mesilate, a synthetic protease inhibitor with anticoagulant properties, is useful for the treatment of adult respiratory distress syndrome, we examined its effect on endotoxin-induced pulmonary vascular injury in rats. DESIGN Prospective, randomized, controlled study. SETTING Research laboratory at a university medical center. SUBJECTS Male Wistar rats (180 to 220 g.) INTERVENTIONS Animals received intravenous infusions of endotoxin (5 mg/kg iv) or saline (control). Pulmonary vascular injury was assessed 6 hrs after administration of endotoxin in terms of the increase in vascular permeability. Rats received gabexate mesilate (10 mg/kg ip), heparin, antithrombin III, an inactive derivative of activated factor X (a selective inhibitor of thrombin generation), or N-[2-[4-(2,2-dimethyl-propionyloxy) phenylsulfonylamino] benzoyl] aminoacetic acid (ONO-5046) (a potent granulocyte elastase inhibitor) 30 mins before endotoxin administration. Leukocytopenia was induced by administration of methotrexate. The effects of the gabexate mesilate on the function of activated neutrophils and the production of tumor necrosis factor -alpha (TNF-alpha) by endotoxin-stimulated monocytes were examined in vitro using neutrophils and monocytes prepared from healthy human volunteers. MEASUREMENTS AND MAIN RESULTS Pulmonary vascular permeability was determined by measuring the vascular leakage of intravenously administered 125I-labeled bovine serum albumin. Intravenous administration of endotoxin significantly increased pulmonary vascular permeability. Gabexate mesilate significantly inhibited pulmonary vascular injury observed 6 hrs after the administration of endotoxin. Pulmonary vascular injury was not attenuated by the administration of heparin, heparin plus antithrombin III, or the inactive derivative of activated factor X, but pulmonary vascular injury was significantly attenuated in animals with methotrexate-induced leukocytopenia and in those animals treated with N-[2-[4-(2,2-dimethyl-propionyloxy) phenylsulfonylamino] benzoyl] aminoacetic acid. Gabexate mesilate in concentrations of 10(-4) to 10(-3) M inhibited the release of granulocyte elastase and leukocyte aggregation stimulated by N-formyl-methionyl-leucyl-phenylalanine and the opsonized zymosan-activated production of superoxide radical by neutrophils in vitro. Gabexate mesilate significantly inhibited the endotoxin-induced increase in the serum concentration of TNF-alpha in vivo and, at a concentration of 10(-8) M, the production of TNF-alpha by endotoxin-stimulated monocytes in vitro. CONCLUSION Our findings suggest that gabexate mesilate attenuated endotoxin-induced pulmonary vascular injury mainly by inhibiting TNF-alpha production by monocytes, which may play a central role in sepsis-related lung injury.