Kevin M. Kadesky

Intestinal reperfusion up-regulates inducible nitric oxide synthase activity within the lung*

BACKGROUND This study examines the hypothesis that pulmonary inducible nitric oxide synthase (iNOS) activity is up-regulated during intestinal reperfusion and that inhibition of NO generation exacerbates pulmonary microvascular dysfunction. METHODS Sprague-Dawley rats underwent intestinal ischemia and reperfusion (IIR) or sham operation (SHAM). Pulmonary iNOS activity was measured by quantitating the conversion of L-arginine (L-Arg) to L-citrulline. Another set of animals undergoing IIR or SHAM received an inhibitor of NOS (NG-nitro-L-arginine methylester; L-NAME; 20 mg/kg intravenously), substrate for NO generation (L-Arg; 300 mg/kg intravenously), or vehicle (normal saline solution; 3 ml). Pulmonary microvascular dysfunction was then quantitated by measuring the extravasation of Evans blue dye (EBD) into the lung. RESULTS Inducible NOS activity was six times greater in the lungs of animals sustaining IIR when compared with SHAM (p = 0.0005). The concentration of EBD within the lungs of animals sustaining IIR was 30% greater than SHAM (p < 0.05). Inhibiting NOS with L-NAME significantly increased pulmonary EBD concentration of both IIR and SHAM groups when compared with normal saline solution-treated animals (p < 0.0001). Treatment with L-Arg prevented this IIR-induced increase in pulmonary dye extravasation. CONCLUSIONS These data suggest that pulmonary iNOS activity is up-regulated in animals sustaining IIR and that this may serve as a compensatory protective response to remote organ injury.

Hepatic hypoperfusion after intestinal reperfusion

BACKGROUND Intestinal ischemia-reperfusion injury (IIR) induces hepatic and pulmonary dysfunction and thus has been used as a model of multiple organ failure syndrome. This study examines the hypothesis that hepatic blood flow is markedly reduced in this injury model. METHODS Sprague-Dawley rats underwent 120 minutes of intestinal ischemia and 60 minutes of reperfusion (IIR). Hepatic blood flow was measured with radiolabeled microspheres and Doppler flow probes. Hepatic dysfunction was quantitated by measuring bile flow and serum alanine aminotransferase and hepatic tissue adenosine triphosphate levels. Sham-operated animals served as controls. RESULTS Intestinal ischemia reduced portal flow by 66% when compared with sham-operated animals (p = 0.0001) but had no effect on hepatic arterial flow. In contrast, reperfusion reduced hepatic artery flow by 80% when compared with controls (p = 0.002) with most of this change occurring within 5 minutes of reperfusion. IIR induced a 63% reduction in bile flow (p < 0.05), a fivefold rise in serum alanine aminotransferase level (p < 0.0002), and a 33% reduction in hepatic adenosine triphosphate level (p < 0.05). CONCLUSIONS These data suggest that IIR induces profound hepatic hypoperfusion, which is temporally related to acute hepatic dysfunction. This observation suggests that hepatic ischemia may contribute to IIR-induced liver injury.

Neutrophil regulation of splanchnic blood flow after hemorrhagic shock

ObjectiveThis study examines the hypothesis that neutrophils impair splanchnic blood flow during resuscitation from hemorrhage by inhibiting the release of the compensatory vasodilator PGl2 from the bowel. Summary Background DataResuscitation from hemorrhagic shock is associated with neutrophil infiltration into the intestine, reduced splanchnic perfusion, and reduced release of PGl2 from the intestine. MethodsSprague-Dawley rats received either vinblastine (VIN) to deplete circulating neutrophils or normal saline (NS). These animals then underwent either hemorrhage and resuscitation (SK + R) or sham operation (SHAM). Superior mesenteric artery flow and splanchnic 6-keto PGF1a (metabolite of PGl2) release were measured. ResultsSuperior mesenteric artery blood flow was significantly greater in VIN-treated animals sustaining SK + R than in those treated with NS (p < 0.05). Neutrophil depletion preserved 6-keto PGF1a release after SK + R, whereas 6-keto PGF1a release in the NS-treated, SK + R group was significantly reduced (p < 0.05). ConclusionThese data are compatible with the hypothesis that neutrophils may influence splanchnic perfusion after SK + R by inhibiting splanchnic PGl2 release.

In vitro evidence of neutrophil-mediated lung injury after intestinal reperfusion.

ABSTRACT This study examines the hypothesis that neutrophils isolated from animals sustaining intestinal reperfusion (IIR) induce pulmonary microvascular dysfunction. Lungs were isolated from normal Sprague-Dawley rats and perfused with a physiologic buffer in vitro. Neutrophils (2 × 106) isolated from animals sustaining IIR (n = 5) or sham operation (SHAM; n = 6) were infused into the isolated lung model. A third group of lungs underwent in vitro perfusion without exposure to neutrophils (n = 5). Lung injury was assessed by measuring wet to dry weight ratios and pulmonary artery pressure (PAP). Pulmonary ultrastructure was assessed by electron microscopy. The wet:dry ratio of lungs from animals sustaining IIR was greater than that of lungs exposed to SHAM neutrophils (p = .03) or perfusate alone (p = .02). The PAP of lungs exposed to IIR neutrophils was nearly 10 times greater than that of lungs exposed to SHAM neutrophils (p = .003) or buffer alone (p = .006). Ultrastructural examination of lungs exposed to IIR neutrophils demonstrated interstitial edema with occasional focal disruptions in the alveolar capillary endothelial cell membrane whereas lungs exposed to SHAM neutrophils were normal. These experiments provide important in vitro correlation of prior in vivo studies suggesting that neutrophils are important pathogenic mediators of IIR-induced lung injury.

Platelet activating factor (PAF) stimulates release of PGI2 from inflamed rabbit gallbladder cell cultures

This study examines the hypothesis that PAF stimulates release of PGI2 from inflamed rabbit gallbladder explant cell cultures. New Zealand white rabbits underwent bile duct ligation for 72 h (72 h BDL), or sham operation, Sham and 72 h BDL gallbladder explants were placed in culture, and the cells grown to 75% confluence. The cells were exposed to increasing concentrations of PAF for 60 min. The media analyzed for eicosanoid release by EIA and the cells analyzed for cyclooxygenase and prostacyclin synthase content by immunoblot analysis. PAF increased release of 6-keto-PGF1 alpha from the 72 h BDL gallbladder cell cultures in a dose-related manner which was inhibited by indomethacin preincubation by 90%. The increased 72 h BDL cell release of 6-keto-PGF1 alpha was not associated with changes in the content of cyclooxygenase or prostacyclin synthase. PAF did not alter eicosanoid release from sham control cell cultures. These data suggest that PAF can only up-regulate endogenous 6-keto-PGF1 alpha release from the 72 h BDL cells that had been previously stimulated by inflammation. PAF may thus contribute to gallbladder distention and injury by chronic stimulation of inflamed gallbladder PGI2 release.

Endotoxic shock after long-term resuscitation of hemorrhage/reperfusion injury decreased splanchnic blood flow and eicosanoid release.

OBJECTIVE The authors examine the hypothesis that hemorrhage/reperfusion injury predisposes the splanchnic bed to decreased prostacyclin (PGl2) release and blood flow after subsequent endotoxin challenge. SUMMARY BACKGROUND DATA Prostacyclin is a potent vasodilator that has been demonstrated to be an important regulator of splanchnic blood flow. Previous studies have demonstrated that during resuscitation from severe hemorrhage, there is a marked reduction in intestinal PGl2 levels, which is associated with reduced splanchnic perfusion. METHODS Anesthetized Sprague-Dawley rats underwent hemorrhage to a mean arterial pressure of 30 mmHg for 30 minutes followed by the reinfusion of shed blood. Then the animals were maintained on total parenteral nutrition (TPN) for 10 days, after which time they received 20 mg/kg Escherichia coli endotoxin intraperitoneally. Aortic and superior mesenteric artery (SMA) blood flow was monitored with a Doppler flow probe. The splanchnic bed was excised and perfused in vitro for measurement of venous effluent eicosanoid concentrations. Controls consisted of animals that received TPN and endotoxin but did not undergo hemorrhage and resuscitation (sham). RESULTS Total parenteral nutrition support of sham animals followed by endotoxin challenge did not alter splanchnic eicosanoid release or blood flow. Hemorrhage/reperfusion animals supported by long-term TPN and challenged with endotoxin demonstrated a threefold decrease in splanchnic prostacyclin metabolite (6-keto-PGF1 alpha) release and a 50% decrease in SMA blood flow. CONCLUSIONS Hemorrhage/reperfusion injury predisposes the splanchnic bed from rats sustained with long-term TPN to decreased release of PGl2 and SMA blood flow when challenged with endotoxin as a second injury.

LONG TERM RESUSCITATION OF HEMORRHAGE/ REPERFUSION INJURY (H/R) STIMULATES RENAL PGE2 RELEASE.: 13

This study examines the hypothesis that long-term resuscitation with hyperalimentation (TPN) following acute hemorrhage/reperfusion (H/R) injury stimulates renal release of PGE2. Male Sprague-Dawley rats were anesthetized and subjected to sham or hemorrhage to 30 mmHg for 30 min followed by reperfusion. All rats were placed on TPN for 5 days, then underwent laparotomy for in vivo renal artery and aortic blood flow for 60 min. The kidney was perfused in vitro with Krebs-Henseleit buffer at 3 ml/min (pH 7.4, 37 degrees C) and venous effluent was collected for analysis of PGE2, 6-keto-PGF1 alpha and thromboxane B2 by EIA. Hemorrhage/reperfusion followed by TPN for 5 days increased renal PGE2 2-fold and decreased in vivo renal artery blood flow by 50% compared to the sham group. Hemorrhage/reperfusion followed by TPN did not alter release of the other eicosanoids measured. These data suggest that the kidney has a limited capacity to maintain renal blood flow by increasing release of PGE2 when the animal is subjected to long-term resuscitation with TPN following mild hemorrhage/reperfusion injury.