Brenda Austin

Human neutrophil activation and increased adhesion by various resuscitation fluids

Objective: To determine whether activated neutrophils play a major role in secondary tissue injury after resuscitation in trauma. We hypothesized that human neutrophil activation and adhesion vary, depending on the type and amount of resuscitation fluid used. Setting: University‐based research facility. Subjects: Ten healthy adult volunteers. Design: Whole blood from volunteers was serially diluted in polypropylene tubes with various resuscitation fluids. Fluids tested were phosphate‐buffered saline, normal saline, lactated Ringers solution, dextran, hespan, 5% human albumin, 25% human albumin, 3.5% hypertonic saline, and 7.5% hypertonic saline. Neutrophil activation (intracellular oxidative burst activity with dichlorofluorescin diacetate staining) and adhesion (integrin cell surface expression of CD18) were measured with flow cytometry (fluorescence‐activated cell sorting). Blood was diluted with hypertonic saline by controlling for sodium content equal to normal saline. Measurements and Main Results: There was a significant dose‐related increase in neutrophil oxidative burst activity as the result of dilution followed with crystalloid fluids and artificial colloids (dextran and hespan). The increase was 12‐18 × baseline at the 75% dilution. The increase with 5% human albumin was only 2.2 × baseline, and 25% albumin did not demonstrate any increased intracellular activity. A similar significant increase in the neutrophil adhesion expression (CD18) occurred with artificial colloids (p < .05) and, to a lesser extent, with crystalloids, but not with albumin. Hypertonic saline caused a decrease in CD18 cell surface expression. Conclusions: This study suggests that the neutrophil activation and adhesion may vary, depending on the type of resuscitative fluid used. All artificial resuscitative fluids may not be similar or innocuous, as demonstrated by the dose‐related increase in neutrophil activation and adhesion.

Lactated Ringer's solution resuscitation causes neutrophil activation after hemorrhagic shock

PURPOSE To determine the degree of neutrophil activation caused by hemorrhagic shock and resuscitation. METHODS Awake swine underwent 15-minute 40% blood volume hemorrhage, and a 1-hour shock period, followed by resuscitation with: group I, lactated Ringers solution (LR); group II, shed blood; and group III, 7.5% hypertonic saline (HTS). Group IV underwent sham hemorrhage and LR infusion. Neutrophil activation was measured in whole blood using flow cytometry to detect intracellular superoxide burst activity. RESULTS Neutrophil activation increased significantly immediately after hemorrhage, but it was greatest after resuscitation with LR (group I, 273 vs. 102%; p < 0.05). Animals that received shed blood (group II) and HTS (group III) had neutrophil activity return to baseline state after resuscitation. Group IV animals had an increase in neutrophil activation (259 vs. 129%; p < 0.05). CONCLUSION Neutrophil activation occurring after LR resuscitation and LR infusion without hemorrhage, but not after resuscitation with shed blood or HTS, suggests that the neutrophil activation may be caused by LR and not by reperfusion.

Controlled resuscitation for uncontrolled hemorrhagic shock.

OBJECTIVE To test the hypothesis that controlled resuscitation can lead to improved survival in otherwise fatal uncontrolled hemorrhage. METHODS Uncontrolled hemorrhage was induced in 86 rats with a 25-gauge needle puncture to the infrarenal aorta. Resuscitation 5 minutes after injury was continued for 2 hours with lactated Ringers solution (LR), 7.3% hypertonic saline in 6% hetastarch (HH), or no fluid (NF). Fluids infused at 2 mL x kg(-1) x min(-1) were turned on or off to maintain a mean arterial pressure (MAP) of 40, 80, or 100 mm Hg in six groups: NF, LR 40, LR 80, LR 100, HH 40, and HH 80. Blood loss was measured before and after 1 hour of resuscitation. RESULTS Survival was improved with fluids. Preresuscitation blood loss was similar in all groups. NF rats did not survive 4 hours. After 72 hours, LR 80 rats (80%) and HH 40 rats (67%) showed improved survival over NF rats (0%) (p < 0.05). Rebleeding increased with MAP. Attempts to restore normal MAP (LR 100) led to increased blood loss and mortality. CONCLUSION Controlled resuscitation leads to increased survival compared with no fluids or standard resuscitation. Fluid type affects results. Controlled fluid use should be considered when surgical care is not readily available.

Resuscitation with lactated Ringer's solution in rats with hemorrhagic shock induces immediate apoptosis

BACKGROUND We hypothesize that different resuscitative fluids may immediately affect the degree of apoptosis after hemorrhagic shock. METHODS Rats (n = 35) were hemorrhaged 27 mL/kg over 5 minutes followed by 1 hour of shock, then resuscitation over 1 hour. The six treatment groups were sham hemorrhage, sham resuscitation, whole blood resuscitation, lactated Ringers solution (LR) resuscitation with three times the volume bled, sham hemorrhage with LR infusion, and 7.5% hypertonic saline resuscitation (9.7 mL/kg). Liver and small intestine were harvested immediately after resuscitation. Apoptosis was evaluated by using in situ cell death detection method. RESULTS Resuscitation with LR resulted in a significant increase in small intestinal and liver apoptosis. Animals that received LR infusion without hemorrhage had an increased level of apoptosis in the intestine. Apoptosis in the intestine was observed in both the mucosa and muscularis externa. There was no increase in apoptosis in either organ in the animals resuscitated with sham resuscitation, whole blood, and hypertonic saline compared with the sham hemorrhage group. CONCLUSION Resuscitation with LR solution after hemorrhagic shock increased immediate cell death by apoptosis in both the small intestine and liver. There was no significant increase in apoptosis in the animals resuscitated with hypertonic saline, whole blood, or in unresuscitated animals. Thus, the type of resuscitation fluid used may affect the apoptotic cellular response to shock.

Resuscitation-induced pulmonary apoptosis and intracellular adhesion molecule-1 expression in rats are attenuated by the use of Ketone Ringer's solution.

BACKGROUND Resuscitation with Lactated Ringers solution after hemorrhagic shock in rats has been shown to cause early cellular injury in the lung. We hypothesized that the use of energy substrates, such as ketone bodies, in the resuscitation fluids would protect against this injury. As markers of cellular injury we measured the induction of apoptotic cell death and the expression of Intracellular Adhesion Molecule-1 (ICAM-1). STUDY DESIGN Male Sprague Dawley rats (n = 35) under inhaled isoflurane anesthesia had placement of femoral arterial and venous catheters. A three-stage hemorrhage model was used for this experiment. There was an initial hemorrhage of 27 mL/kg for 10 minutes. During the next 75 minutes another 8 mL/kg of blood was withdrawn at a steady rate. The resuscitation fluids were then infused for 45 minutes during which the third continuous hemorrhage of 8 mL/kg was performed. The animals were randomized to five groups: 1) sham hemorrhage (n = 6); 2) sham resuscitation (n = 7); 3) Lactated Ringers resuscitation, three times the volume of shed blood (n = 8); 4) Ketone Ringers (containing 28 mEq/L of beta-hydroxybutyrate) resuscitation, three times the volume of shed blood (n = 7); and 5) plasma resuscitation, volume equal to shed blood (n = 7). The animals were sacrificed 1 hour after resuscitation and lungs were harvested. Western blot technique was used for the determination of proapoptotic protein (bax), antiapoptotic protein (bcl-2), apoptotic fragments of poly ADP-ribose polymerase, and ICAM-1. Sections of lung were also subjected to immunostaining using antibodies to bax and ICAM-1 proteins (reported as number of positive cells/mm2). RESULTS Lactated Ringers resuscitation caused a significant increase in pulmonary apoptosis and ICAM-1 expression compared with the sham hemorrhage group. Animals resuscitated with Ketone Ringers solution and plasma did not show this injury pattern. CONCLUSIONS Substitution of lactate with ketone bodies in the resuscitation fluid attenuates the expression of cellular injury markers in the lung.