Manuel S. Baird

Evaluation of the contribution of platelets to clot strength by thromboelastography in rabbits: the role of tissue factor and cytochalasin D.

The contribution of platelets and soluble clotting components to clot strength has been the focus of several clinical studies using thromboelastography; it would, therefore, be beneficial to develop an animal model with which to mechanistically approach hemostatic disorders. Thus, we proposed to determine if the contribution of platelet function (GP, dyne/cm2) and soluble components of the coagulation pathway to total clot strength (GT) in rabbits were similar to those in humans. Blood was sampled from the ear arteries of conscious rabbits (n = 12); 350 &mgr;L of the blood was placed in a thromboelastograph. Ten microliters of normal saline, cytochalasin D (an inhibitor of microtubule function, 10 &mgr;M final concentration), or tissue factor (a potent stimulator of platelet function, 0.00625% final concentration) was added to the blood sample, and thromboelastography performed for 1 h. The GT (mean ± sd) was significantly (P < 0.001) different among samples exposed to normal saline, cytochalasin D, or tissue factor, with GT values of 7238 ± 1432, 937 ± 372, and 16,556 ± 3314, respectively. GP was responsible for 87% and 94% of GT in the absence or presence of tissue factor, respectively. GP did not significantly correlate with platelet concentration in the absence or presence of tissue factor. The contribution of GP to GT is similar to that observed in humans. Implications Rabbits may serve as a model of hemostasis that closely approximates human situations to mechanistically determine the etiology of coagulopathy. The contribution of platelet function to total clot strength is similar to that observed in humans.

cAMP activation of chloride and fluid secretion across the rabbit alveolar epithelium

Active Na+ transport by alveolar epithelial cells has been demonstrated to contribute significantly to alveolar fluid clearance. However, the contribution of transepithelial Cl- movement to the reabsorption of isosmotic fluid across the alveolar epithelium in vivo has not been elucidated. We hypothesized that Cl- transport could be increased across the alveolar epithelium in vivo and across cultured alveolar type II cells by agents that increase intracellular cAMP (e.g., forskolin). In studies where 5% albumin in sodium methanesulfonate (a Cl--free solution) was administered into the lung, forskolin administration significantly increased intracellular influx of Cl- and fluid into the alveolar space. In vitro studies with cultured rabbit alveolar type II cell monolayers in Ussing chambers demonstrated that elevations in intracellular cAMP increase short-circuit current by increasing both Cl- secretion and Na+ reabsorption. The cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide and the loop diuretic bumetanide partially decreased the forskolin-induced increase in short-circuit current. These data may explain the failure of agonist that stimulated intracellular cAMP to increase alveolar fluid clearance in the rabbit. Moreover, the data suggest that in the event Na+ absorptive pathways are damaged, transepithelial Cl- secretion and the consequent intra-alveolar fluid influx may be upregulated.Active Na+ transport by alveolar epithelial cells has been demonstrated to contribute significantly to alveolar fluid clearance. However, the contribution of transepithelial Cl- movement to the reabsorption of isosmotic fluid across the alveolar epithelium in vivo has not been elucidated. We hypothesized that Cl- transport could be increased across the alveolar epithelium in vivo and across cultured alveolar type II cells by agents that increase intracellular cAMP (e.g., forskolin). In studies where 5% albumin in sodium methanesulfonate (a Cl--free solution) was administered into the lung, forskolin administration significantly increased intracellular influx of Cl- and fluid into the alveolar space. In vitro studies with cultured rabbit alveolar type II cell monolayers in Ussing chambers demonstrated that elevations in intracellular cAMP increase short-circuit current by increasing both Cl-secretion and Na+ reabsorption. The cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide and the loop diuretic bumetanide partially decreased the forskolin-induced increase in short-circuit current. These data may explain the failure of agonist that stimulated intracellular cAMP to increase alveolar fluid clearance in the rabbit. Moreover, the data suggest that in the event Na+absorptive pathways are damaged, transepithelial Cl- secretion and the consequent intra-alveolar fluid influx may be upregulated.

Hextend (hetastarch solution) decreases multiple organ injury and xanthine oxidase release after hepatoenteric ischemia-reperfusion in rabbits.

OBJECTIVE We hypothesized that multiple organ injury and concentrations of xanthine oxidase (an oxidant-generating enzyme released after hepatoenteric ischemia) would be decreased by the administration of a bolus of a colloid solution at reperfusion. DESIGN Randomized, masked, controlled animal study. SETTING University-based animal research facility. SUBJECTS Fifty-four New Zealand white male rabbits, weighing 2 to 3 kg. INTERVENTIONS Anesthetized rabbits were assigned to either the hepatoenteric ischemia-reperfusion group (n = 27) or the sham-operated group (n = 27). Hepatoenteric ischemia was maintained for 40 mins with a balloon catheter in the thoracic aorta, followed by 3 hrs of reperfusion. Each group was randomly administered a bolus of one of three fluids at the beginning of reperfusion: Hextend (hetastarch solution); 5% human albumin; or lactated Ringers solution. The investigators were masked as to the identity of the fluid administered. MEASUREMENTS AND MAIN RESULTS Multiple organ injury was assessed by the release of lactate dehydrogenase activity into the plasma and by indices of gastric and pulmonary injury. Circulating lactate dehydrogenase activity was significantly greater (p < .001) in animals receiving lactated Ringers solution than in rabbits receiving either colloid solution. Gastric injury (tissue edema, Histologic injury Score) was significantly decreased (p < .01) by administration of both colloid solutions. Lung injury (bronchoalveolar lavage lactate dehydrogenase activity) was significantly decreased (p < .05) by the hetastarch solution administration. The hetastarch solution administration resulted in 50% less xanthine oxidase activity release during reperfusion compared with albumin or lactated Ringers solution administration (p < .001). CONCLUSION We conclude that multiple organ injury and xanthine oxidase release after hepatoenteric ischemia-reperfusion are decreased by colloid administration.

Gastric intramucosal pH and multiple organ injury: impact of ischemia-reperfusion and xanthine oxidase.

OBJECTIVES To determine if gastric intramucosal pH is affected by hepatoneteric ischemia-reperfusion. We additionally proposed to determine if changes in gastric mucosal hydrogen ion concentration are associated with liver and lung injury following hepatoenteric ischemia-reperfusion. Finally, we hypothesized that gastric intramucosal pH is influenced by xanthine oxidase, an oxidant-generating enzyme released after hepatoenteric ischemia-reperfusion. DESIGN Randomized, controlled, animal study. SETTING University-based animal research facility. SUBJECTS Thirty-six New Zealand white male rabbits (2 to 3 kg). INTERVENTIONS Anesthetized rabbits were randomly assigned to one of four groups (n = 9 per group): a) sham-operated group; b) sham-operated group pretreated with sodium tungstate (xanthine oxidase inactivator); c) aorta occlusion group; and d) aorta occlusion group pretreated with sodium tungstate. Descending thoracic aorta occlusion was maintained for 40 mins with a 4-Fr Fogarty embolectomy catheter, followed by 2 hrs of reperfusion. MEASUREMENTS AND MAIN RESULTS Gastric tonometry was performed after completion of the surgical preparation (30-min equilibration) and at 30, 60, 90, and 120 mins of reperfusion. Plasma alanine aminotransferase activity was determined at 120 mins of reperfusion to assess hepatic injury. Bronchoalveolar lavage of the right lung was performed after 120 mins of reperfusion, and the protein content was determined as a measure of pulmonary alveolar-capillary membrane compromise. Descending thoracic aorta occlusion resulted in a significant decrease in gastric intramucosal pH as compared with sham-operated rabbits (p < .001). The change in gastric mucosal hydrogen ion concentration was significantly associated with plasma alanine aminotransferase activity (r2 = .48, p < .01) and bronchoalveolar protein content (r2 = .51, p < .01). Xanthine oxidase inactivation significantly improved gastric intramucosal pH after aortic occlusion and reperfusion (p < .001), with a concomitant attenuation of the release of plasma alanine aminotransferase (p < .05) and accumulation of bronchoalveolar protein (p < .05) during reperfusion. CONCLUSIONS Gastric intramucosal pH was significantly decreased after hepatoenteric ischemia-reperfusion. Furthermore, an increase in gastric intramucosal hydrogen ion concentration was associated with a concomitant increase in tissue injury, a presumed harbinger of multiple organ failure. Gastric intramucosal pH values improved during reperfusion after xanthine oxidase inactivation, concomitant with attenuation of hepatic and pulmonary injury. Gastric tonometry is an important clinical tool that can provide critical insight into the pathogenesis of multiple organ injury after hepatoenteric ischemia-reperfusion. Gastric tonometry may aid in the rapid assessment of pharmacologic interventions designed to attenuate multiple organ injury in similar clinical settings (e.g., trauma, shock, major vascular surgery).

Xanthine oxidase mediates myocardial injury after hepatoenteric ischemia-reperfusion

OBJECTIVES To determine if myocardial injury results from hepatoenteric ischemia-reperfusion. We also proposed to determine if this remote heart injury is mediated by a xanthine oxidase-dependent mechanism. DESIGN Randomized, controlled animal study. SETTING University-based animal research facility. SUBJECTS Thirty-six New Zealand white male rabbits, weighing 1.8 to 3 kg. INTERVENTIONS Anesthetized rabbits were randomly assigned to one of four groups (n = 9 per group): a) a sham-operated group; b) a sham-operated group pretreated with sodium tungstate (xanthine oxidase inactivator); c) an aorta occlusion group; and d) an aorta occlusion group pretreated with sodium tungstate. Descending thoracic aorta occlusion was maintained for 40 mins with a 4-Fr Fogarty embolectomy catheter, followed by 2 hrs of reperfusion. MEASUREMENTS AND MAIN RESULTS Myocardial injury, manifested by increased circulating creatine kinase-MB fraction activity, was significantly associated with aortic occlusion and reperfusion (p < .05). Sodium tungstate pretreatment significantly (p < .05) reduced circulating and myocardial xanthine oxidase activity. Xanthine oxidase inactivation by sodium tungstate significantly decreased circulating creatine kinase-MB fraction activity after hepatoenteric ischemia-reperfusion (p < .05). Finally, circulating creatine kinase-MB fraction activity was significantly associated with circulating xanthine oxidase activity (r2 = .85; p < .001). CONCLUSIONS We conclude that remote myocardial injury is caused by hepatoenteric ischemia-reperfusion. The pathoetiology of this myocardial injury involves a xanthine oxidase-dependent mechanism.

Extreme hemodilution in rabbits: an in vitro and in vivo Thrombelastographic analysis.

Isovolemic hemodilution is used to decrease the incidence of blood transfusions. However, the effects of the degree of hemodilution and the fluid used on hemostasis are controversial. We tested the hypothesis that hemodilution and the fluid administered would adversely alter Thrombelastographic® (Haemoscope, Skokie, IL) variables (reaction time, &agr; angle and maximal amplitude). Conscious rabbits had blood sampled from ear arteries and diluted 0% or 75%in vitro with one of four solutions: 6% hetastarch in 0.9% NaCl, 5% human albumin in 0.9% NaCl, or balanced electrolyte solutions containing either 6% pentastarch or 6% hetastarch. Isoflurane-anesthetized rabbits were randomly assigned to groups (n = 9 per group) that underwent in vivo isovolemic hemodilution (75% of estimated blood volume removed), with blood replaced with one of the four solutions mentioned previously. In vitro hemodilution resulted in a significant (P < 0.05) decrease in hemostatic function (increase in reaction time, decrease in &agr; angle and maximal amplitude) that was largest after hemodilution with albumin. However, although in vivo hemodilution significantly (P < 0.05) decreased reaction time, increased the &agr; angle, and decreased maximal amplitude, there were no significant fluid-dependent effects. Implications: The effects of hemodilution and the fluid used on Thrombelastographic® (Haemoscope, Skokie, IL) variables are markedly different between in vitro and in vivo hemodilution studies.

Desflurane increases pulmonary alveolar-capillary membrane permeability after aortic occlusion-reperfusion in rabbits: evidence of oxidant-mediated lung injury.

Background Pulmonary injury occurs after vascular surgery, with xanthine oxidase (an oxidant generator) released from reperfusing liver and intestines mediating a significant component of this injury. Because halogenated anesthetics have been observed to enhance oxidant‐mediated injury in vitro, the authors hypothesized that desflurane would increase alveolar‐capillary membrane permeability mediated by circulating xanthine oxidase after thoracic occlusion and reperfusion. Methods Rabbits were assigned to one of five groups: aorta occlusion groups administered desflurane (n = 14), desflurane and tungstate (xanthine oxidase inactivator, n = 12), fentanyl plus droperidol (n = 13), and two sham‐operated groups (desflurane, n = 7 and fentanyl plus droperidol, n = 7). Aortic occlusion was maintained for 45 min with a balloon catheter, followed by 3 h of reperfusion. Alveolar‐capillary membrane permeability was assessed by measurement of bronchoalveolar lavage fluid protein. Xanthine oxidase activity was determined in plasma and lung tissue. Ascorbic acid content (an antioxidant) was determined in lung tissue. Results Desflurane was associated with significantly increased alveolar‐capillary membrane permeability after aortic occlusion‐reperfusion when compared with the fentanyl plus droperidol anesthesia or sham‐operated groups (P < 0.05). Inactivation of xanthine oxidase abrogated the alveolar‐capillary membrane compromise associated with desflurane. Although significantly greater than for sham‐operated animals, plasma xanthine oxidase activities released after aortic occlusion‐reperfusion were not different between the two anesthetics. There were no anesthetic‐associated differences in lung tissue xanthine oxidase activity. However, desflurane anesthesia resulted in a significant reduction in lung ascorbic acid after aortic occlusion‐reperfusion compared with the sham‐operated animals. Conclusions Desflurane anesthesia increased xanthine oxidase‐dependent alveolar‐capillary membrane compromise after aortic occlusion‐reperfusion in concert with depletion of a key tissue antioxidant.

Extreme, Progressive Isovolemic Hemodilution with 5% Human Albumin, PentaLyte, or Hextend Does Not Cause Hepatic Ischemia or Histologic Injury in Rabbits

BACKGROUND Physicians and their patients are greatly concerned about perioperative blood administration. Although isovolemic hemodilution is utilized to decrease the incidence of transfusion, it is unclear at what degree of hemodilution hepatoenteric ischemia and injury occurs. The authors hypothesized that hepatic ischemia, systemic ischemia, and tissue injury would occur during hemodilution in rabbits, and that the severity of ischemia and injury may be dependent on the fluid administered. METHODS Rabbits anesthetized with isoflurane were assigned randomly to a sham-operated group (n = 8) or groups that underwent four isovolemic hemodilutions (25% of the blood volume removed at hourly intervals), with blood replaced with one of three solutions: balanced electrolyte solutions containing 6% pentastarch (n = 8), 6% hetastarch (n = 9), or 5% human albumin in normal saline (n = 8). Arterial ketone body ratio and plasma lactate, respectively, served as measures of hepatic and systemic ischemia. Gastric, duodenal, and hepatic histologic injury was assessed post mortem. RESULTS Hemodilution from a baseline hematocrit of about 33% to about 8% (third hemodilution) with all three colloids did not result in a significant increase in plasma lactate concentration or decrease in arterial ketone body ratio. At a hematocrit of about 5% (fourth hemodilution), the hetastarch group had a significantly (P < 0.05) greater plasma lactate concentration than the sham-operated and 5% human albumin groups. There were no significant differences in arterial ketone body ratio or histologic injury between the groups. CONCLUSIONS Isovolemic hemodilution (approximately 5% hematocrit) with albumin, pentastarch, or hetastarch solutions does not result in significant hepatic ischemia or injury assessed by histology.

halothane and Xanthine Oxidase Increase Hepatocellular Enzyme Release and Circulating Lactate after Ischemia-reperfusion in Rabbits

Background: Multiple‐organ injury often occurs after aortic occlusion‐reperfusion. Oxidants derived from xanthine oxidase have been implicated as a source of injury after aortic occlusion‐reperfusion. Halogenated anesthetics modify oxidant‐mediated injury. The current study determined if halothane modifies hepatocellular enzyme release (e.g., alanine aminotransferase) and circulating lactate after aortic occlusion‐reperfusion. Methods: Rabbits were randomly assigned to one of four groups that underwent 40 min of thoracic aortic occlusion and 2 h of reperfusion: Two groups were given either halothane or fentanyl plus droperidol anesthesia and two groups were given either anesthetic and sodium tungstate (xanthine oxidase inactivator). Each of the four groups was then matched with a similarly treated group that did not undergo aortic occlusion. Results: Halothane anesthesia was associated with significantly (P < 0.05) increased release of alanine aminotransferase (34 +/‐ 9 U/l at baseline and 539 +/‐ 370 U/l at 120 min of reperfusion; mean +/‐ SD) and increased plasma lactate concentrations (2.8 +/‐ 2.0 mM at baseline and 12.1 +/‐ 9.7 mM at 120 min of reperfusion) after aortic occlusion‐reperfusion compared with fentanyl plus droperidol anesthesia (alanine aminotransferase, 33 +/‐ 12 U/l and 148 +/‐ 109 U/l; lactate, 3.4 +/‐ 2.0 mM and 3.8 +/‐ 1.2 mM at baseline and 120 min of reperfusion, respectively). Inactivation of xanthine oxidase significantly decreased the release of hepatocellular enzymes (P < 0.05) and decreased circulating lactate in animals anesthetized with halothane after aortic occlusion‐reperfusion. Conclusions: Halothane increased hepatocellular enzyme release and circulating lactate after aortic occlusion‐reperfusion compared with fentanyl plus droperidol anesthesia. Xanthine oxidase activity inactivation also decreased hepatocellular enzyme activity release during reperfusion. These findings justify further investigations to determine if halogenated anesthetics modify tissue injury in clinical settings involving oxidant stress.

Halothane does not decrease amiloride-sensitive alveolar fluid clearance in rabbits.

Halothane decreases alveolar fluid clearance (AFC), a function required for efficient gas exchange in the rat. Further, halothane decreases amiloride-sensitive Na+ transport in rat alveolar type II cells, a process responsible for a significant portion of AFC. We tested the hypothesis that halothane would decrease amiloride-sensitive AFC in rabbits. Rabbits anesthetized with 1.8% halothane had 5% albumin in 0.9% NaCl instilled into the right lung with (n = 11) or without (n = 11) 1 mM amiloride present in the instillate. Similarly, animals anesthetized with IV fentanyl and droperidol were administered 5% albumin solution with (n = 11) or without (n = 11) amiloride. At 90 min after instillation, alveolar fluid samples were obtained, and AFC was determined by changes in fluid protein concentration. Rabbits anesthetized with halothane or fentanyl and droperidol in the absence of amiloride had similar AFC values (35% ± 12% and 35% ± 7%, respectively, mean ± sd). Rabbits anesthetized with halothane or fentanyl and droperidol in the presence of amiloride had similar AFC values (20% ± 10% and 16% ± 12%, respectively) that were significantly less than the groups not administered amiloride (P < 0.01). Unlike the rat, the ability of the rabbit to clear fluid from the alveolar space through amiloride-sensitive pathways is not decreased by halothane anesthesia. Implications Unlike the rat, the ability of the rabbit to clear fluid from the alveolar space through amiloride-sensitive pathways is not decreased by halothane anesthesia.