Lisa L. Trenthem

Reduced tumor necrosis factor production in endotoxin-spiked whole blood after trauma: Experimental results and clinical correlation

BACKGROUND The overproduction of tumor necrosis factor-alpha (TNF) plays a key role in virtually every experimental model of septic shock, which has led to the development of several therapies that target TNF and other cytokines in clinical sepsis. However, our previous work showed that plasma TNF was reduced, rather than increased, when a septic challenge was administered 3 days after hemorrhagic shock. In this study we compared whole-blood TNF production ex vivo in human beings and animals after trauma. METHODS TNF was measured before and after a 4-hour incubation of whole blood with 0 or 5 micrograms/ml Escherichia coli endotoxin (LPS) at 37 degrees C ex vivo. Samples were obtained from trauma patients with (n = 8) and without (n = 14) sepsis and compared with those obtained in healthy volunteers (n = 11). In parallel experiments in a pig model TNF was measured before and after fluid resuscitation from trauma after an ex vivo (0 or 5 micrograms/ml LPS) or an in vivo (5 micrograms/kg LPS, 30 minutes intravenously) challenge. RESULTS With either an immunoassay or a bioassay in either human beings or pigs before or after trauma, TNF was at or below the threshold of detection, unless the blood sample was spiked with LPS. After spiking, TNF was markedly elevated, but the increment was reduced after trauma. In pigs an LPS challenge in vivo delayed 3 days after trauma evoked an increment in plasma TNF that was blunted compared with that in an uninjured control. This trauma-induced reduction in blood TNF could not be attributed to a simple reduction in the number of monocytes nor to changes in cortisol, nor to increased numbers of neutrophils, whose proteolytic enzymes can impair production or increase the degradation of TNF. Although the plasma concentration of soluble TNF-binding proteins (60 kd) was elevated in nonsepsis (p = 0.0358) and sepsis trauma patients (p = 0.0148), the correlation with TNF production was relatively weak (R2 = 0.260). CONCLUSIONS There was no evidence of TNF overproduction in whole blood after trauma. If these results could be generalized to other tissues, it would be difficult to justify therapeutic targeting of TNF in exaggerated inflammatory response (or septic complications) after trauma.

Endogenous adenosine and secondary injury after chest trauma.

BACKGROUND No previous studies have examined actions of adenosine or related compounds after blunt chest trauma, but we have shown that the prototype adenosine-regulating agent, acadesine (aminoimidazole carboxamide ribonucleotide [AICAR]), has multiple favorable anti-inflammatory actions after other forms of trauma, ischemia, hemorrhage, and sepsis; and that a progressive inflammatory response in the contralateral (uninjured) lung after unilateral blunt chest trauma is caused (in part) by activation and sequestration of circulating leukocytes (white blood cells [WBCs]). Thus, we hypothesized that AICAR would ameliorate WBC-dependent, secondary pathophysiologic changes after blunt chest trauma. METHODS Mongrel pigs (28+/-1 kg, n = 21) were anesthetized, mechanically ventilated, and injured on the right chest (pulmonary contusion) with a captive bolt gun. Either AICAR (1 mg/kg + 0.2 mg/kg/min) or its saline vehicle were administered for a 12-hour period, beginning 15 minutes before injury. RESULTS Injury caused a three- to fourfold increase in bronchoalveolar lavage (BAL) WBC counts, 10- to 20-fold increases in BAL protein, and 200% increases in lung edema as measured by wet-dry ratio (all p < 0.05), in both the injured (right) and the noninjured (left) lungs. With AICAR versus saline, BAL WBC counts, lung myeloperoxidase levels, and systemic hemodynamics were similar. However, the increases in BAL protein were attenuated by 30% to 50% (p < 0.14, NS) and edema was reduced (p < 0.05) in both lungs. Furthermore, oxygenation, hypercapnia, acidosis (all p < 0.05), and survival were improved (9 of 10 vs. 4 of 11, p < 0.04). CONCLUSION Pretreatment with AICAR before experimental pulmonary contusion ameliorates the trauma-induced destruction of the alveolar capillary membrane, and attenuates the delayed secondary injury in the contralateral uninjured lung, by a mechanism that may be independent of leukocytes. Endogenous adenosine could have a role in the pathophysiologic response after blunt chest injury, with potential sites of action including the endothelium and alveolar macrophage. Adenosine-regulating agents may have therapeutic potential after blunt chest injury, but further studies are needed in clinically relevant models, with administration begun at the time of resuscitation.

Granulocyte colony-stimulating factor and neutrophil-related changes in local host defense during recovery from shock and intra-abdominal sepsis☆☆☆

BACKGROUND We have reported that treatment with exogenous granulocyte colony-stimulating factor (G-CSF) improves abscess localization and reduces mortality without aggravating neutrophil (PMN)-mediated reperfusion injury in a model of septic abdominal trauma. The purpose of this study was to determine actions of G-CSF on PMN function in the peritoneum. METHODS Anesthetized swine were pretreated with broad-spectrum antibiotics and underwent cecal ligation and incision and 35% hemorrhage (trauma). After 1 hour they were resuscitated with shed blood, crystalloid, and either G-CSF (n = 10) or saline solution vehicle (n = 9). The animals were observed for 72 hours. RESULTS After trauma, saline solution treatment increased PMN infiltration into the peritoneum within 2 hours (P = .035), increased peritoneal PMN elastase production (i.e., cytotoxicity) by 24 hours (P = .004), and decreased adherence of peritoneal PMNs to an artificial substrate from 4 to 72 hrs (P = .043). The mean autopsy score was 7.0 +/- 0.5. With G-CSF treatment peritoneal neutrophilia was enhanced (maximum 48 hours, P = .002) and PMN cytotoxicity was augmented and delayed (maximum 48 hours, P = .004). Despite these changes, adherence of peritoneal PMNs was not significantly changed and there was no evidence for PMN-mediated damage in the lung as judged by bronchoalveolar lavage protein, bronchoalveolar lavage PMNs, lung tissue myeloperoxidase, or histologic changes. The mean autopsy score was improved to 4.1 +/- 0.3 (P < .001). CONCLUSIONS G-CSF in resuscitation fluids improved localization of an intra-abdominal septic focus by increased production of circulating PMNs, increased PMN extravasation into the peritoneal cavity, and increased PMN cytotoxicity at the abdominal septic focus, without exaggerating PMN-dependent reperfusion injury in the lung. Therefore these data further support the idea that G-CSF in resuscitation fluids might reduce septic complications in the multiply injured trauma patient.

Acadesine during fluid resuscitation from shock and abdominal sepsis

OBJECTIVE To determine properties of acadesine, the prototype adenosine regulating agent, in an experimental model in which abdominal sepsis is superimposed onto hemorrhagic shock. DESIGN Randomized, blinded animal study. SETTING University-based animal research facility. SUBJECTS Twenty-eight anesthetized mongrel pigs (35.5 +/- 1.1 kg). INTERVENTIONS The cecum was ligated and punctured to produce abdominal sepsis. To produce hemorrhagic shock, 45% to 47% of the estimated blood volume was withdrawn. After 1 hr, shed blood plus supplemental crystalloid (twice the shed blood volume) plus either acadesine (5 mg/kg bolus + 1 mg/kg x 60 min, n = 10) or its vehicle (n = 10) was administered. All animals were awakened and observed for 48 hrs. At 48 hrs, cardiac function, bacterial cultures from the septic focus, and inflammatory changes in the abdomen were quantified. MEASUREMENTS AND MAIN RESULTS After resuscitation with acadesine vs. vehicle, we observed the following: a) arterial blood pressure and cardiac filling pressures were similar but cardiac index, systemic oxygen delivery, and systemic oxygen consumption were increased; b) plasma lactate was higher, systemic vascular resistance was lower, but ileal mucosal blood flow was not measurably altered; c) lipopolysaccharide-evoked tumor necrosis factor production in whole blood ex vivo was reduced; d) in those animals that survived 48 hrs (10/10 vs. 8/10), sepsis-induced cardiac depression, amount of free intraperitoneal fluid, extra abscess inflammatory reaction, abscess wall formation, abscess bacterial counts, and peritoneal bacterial counts, were all similar, but blood bacterial counts were higher. CONCLUSIONS Fluid resuscitation with acadesine produced no adverse hemodynamic consequences and probably improved washout of metabolites from the reperfused microcirculation in sites other than the small intestine or heart. Taken together, these observations suggest that adenosine regulating agents might have therapeutic potential during fluid resuscitation from trauma. However, at least in these extreme conditions, the acute salutary effects of acadesine were probably overwhelmed by polymicrobial sepsis. Further studies must determine whether supplemental adjuvants to boost host defense during recovery from trauma will optimize adenosine-based resuscitation solutions.

Gastric and extragastric actions of the histamine antagonist ranitidine during posttraumatic sepsis

BACKGROUND Histamine H2 antagonists (e.g., ranitidine) are generally thought to specifically reduce gastric acid secretion and are commonly used for stress ulcer prophylaxis in critically ill patients because of their efficacy and safety profile. A few reports suggest that ranitidine might also bind to extragastric sites and/or act as an immunomodulator. The potential effects on posttraumatic sepsis are unknown. METHODS Mongrel pigs (n = 24) were anesthetized with fentanyl, injured by a 10 kg steel bar dropped from a height of 1 m onto the fleshy portion of the posterior thigh, and then 35% of their blood volume was drained through the arterial catheter. All the shed blood plus two times the hemorrhage volume as lactated Ringers solution was infused after a 1-hour shock period. Either vehicle or ranitidine (1.5 mg/kg) was intravenously administered at the time of resuscitation and every 12 hours thereafter in a blinded fashion. After 72 hours a septic challenge was administered (15 micrograms/kg Escherichia coli lipopolysaccharide [LPS] x 30 min). Serial gastroscopy, gastric pH, hemodynamics, leukocyte counts, cortisol, and tumor necrosis factor were recorded for 180 minutes after LPS. RESULTS Immediately before LPS all hemodynamic variables were identical between treatments, but gastric pH was slightly higher and stress gastritis was marginally lower with ranitidine. LPS caused profound leukopenia and a hyperdynamic circulatory response (i.e., tachycardia, increased cardiac output, and decreased peripheral vascular resistance at relatively constant blood pressure); these changes were not altered by ranitidine. Gastric pH remained elevated after LPS with ranitidine, but LPS-induced gastritis was not modified. Ranitidine delayed the LPS-induced ventilation-perfusion imbalance and attenuated the peak increase in the proinflammatory cytokine, tumor necrosis factor, without altering its antiinflammatory opponent, cortisol. Similar changes were observed in four additional animals treated with cimetidine. The proportion of circulating neutrophils and lymphocytes was slightly altered 180 minutes after LPS, but there was no obvious effect on T lymphocytes in vivo, and no effect on the LPS-induced increase in neutrophil CD18 expression in vitro was seen. CONCLUSIONS (1) Ranitidine increased gastric pH, which blunted the stress gastritis caused by trauma but not that caused by LPS; (2) ranitidine delayed the early LPS-evoked pulmonary changes and reduced the tumor necrosis factor spike, which is consistent with a favorable immunomodulatory action that has been reported in patients who are critically ill or are undergoing an elective abdominal surgical procedure; (3) the mechanism is probably related to H2 receptor antagonism rather than to a nonspecific side effect of ranitidine, which suggests that histamine may have a previously unrecognized role in posttraumatic septic responses; and (4) the site of action is probably not in the heart or peripheral resistance vessels, but salutary effects on circulating lymphocytes or neutrophils cannot be excluded.

Plasma tumor necrosis factor and post-traumatic hyperdynamic sepsis evoked by endotoxin.

To examine the role of systemic plasma tumor necrosis factor (TNF) in the septic response following trauma, an endotoxin (lipopolysaccharide (LPS)) challenge was administered to anesthetized mongrel pigs 72 h following either hemorrhagic shock/resuscitation or sham shock. For TNF to be considered a mediator, at least two conditions should be satisfied: a TNF increase should precede other manifestations of the septic response and the magnitude of that increase should correlate with the symptoms. Immediately following resuscitation from shock, hemodynamics were stable, but heart rate, cardiac index (Cl), and systemic oxygen delivery (DO2) were elevated 20–60%, and systemic vascular resistance (SVR) was decreased 40%, relative to the preshock baseline. After 72 h, the animals were reanesthetized, reinstrumented, and all hemodynamic values were near normal in both groups. At this point, either 1.5 (shock, n = 2; sham, n = 2), 15 (shock, n = 7; sham, n = 6) or 150 (shock, n = 11; sham, n = 4) μg/kg of Escherichia coli LPS was administered intravenously over 30 min. Serial hemodynamic data, complete blood counts, and TNF were recorded for 3 h post-LPS. LPS evoked profound leukopenia and pulmonary hypertension within 15 min that was followed by a hyperdynamic septic response (i.e., progressive arterial desaturation, tachypnea, tachycardia, increased Cl, and decreased SVR) and rise in plasma TNF at 60–90 min. In the shock group, LPS-evoked TNF changes were less than or equal to those in the sham group, even though mortality was higher after shock. By 60 min after 15 μg/kg LPS, plasma TNF was 10 ± 2 vs. 21 ± 4 units/ml in shock vs. sham (p < .05). The corresponding mortality after 3 h was 2/7 in shock and 0/6 in sham. After 150 μg/kg LPS, plasma TNF increased to 16–18 units/ml in both groups, but the 3 h mortality was 8/11 in shock and 1/4 in sham. Since plasma TNF did not rise until after other symptoms of an LPS-evoked inflammatory response were already apparent and since the increment in plasma TNF was not potentiated by a prior bout of resuscitated shock, it is unlikely that the response evoked by a septic challenge following traumatic shock can be directly attributed to excessive levels of systemic TNF.

Combination therapy that targets secondary pulmonary changes after abdominal trauma

After abdominal trauma, the lung is susceptible to secondary injury caused by acute neutrophil (PMN) sequestration and alveolar capillary membrane disruption. Adenosine is an endogenous anti-inflammatory metabolite that decreases PMN activation. AICAR ([5-amino-1-[beta-D-ribofuranosyl]imidazole-4-carboxamide]riboside) is the prototype of a novel class of anti-inflammatory drugs that increase endogenous adenosine. After trauma, AICAR administration has been shown to decrease secondary lung injury in models of hemorrhagic shock with delayed lipopolysaccharide challenge and pulmonary contusion. However, early suppression of PMN activation could worsen outcomes after penetrating abdominal trauma. We hypothesized that, after penetrating abdominal trauma, the ideal resuscitation strategy would involve early, short-lived suppression of PMN activation to minimize secondary lung injury, followed by later enhancement of PMN chemotaxis and phagocytosis [using granulocyte colony-stimulating factor (G-CSF)] to lessen late septic complications. G-CSF has not been shown to potentiate PMN mediated pulmonary reperfusion injury. Swine were subjected to cecal ligation/incision and hemorrhagic shock (trauma), followed by resuscitation with shed blood, crystalloid, and either G-CSF, a combination of G-CSF and AICAR, or 0.9% normal saline. At 72 h, bronchoalveolar lavage (BAL) leukocyte counts and protein concentration were determined, and lung tissue analysed for myeloperoxidase (MPO, a measure of PMN infiltration) and microscopic pathology. Analysis of BALs revealed a significant increase protein concentrations and in white blood cell and PMN infiltration (P< 0.05) following trauma. These acute changes were not exacerbated by G-CSF, but were reversed by combined AICAR + G-CSF, which implicates a physiologic role for adenosine. This suggests that combination therapy may have beneficial effects on the lung after trauma.