Tamara L. Berezina

Trauma-hemorrhagic shock mesenteric lymph from rat contains a modified form of albumin that is implicated in endothelial cell toxicity.

It has been proposed that factors originating from the gut after severe trauma/shock are introduced into the systemic circulation through the mesenteric lymphatics and are responsible for the cellular injury and inflammation that culminates in acute multiple organ dysfunction syndrome (MODS). Indeed, it has been shown that lymph collected from shocked but not sham-shocked animals causes endothelial cell death, neutrophil activation, and bone marrow (BM) colony growth suppression in vitro. In an attempt to isolate the factor(s) in lymph responsible for endothelial cell toxicity, lymph from shock and sham animals was fractionated by solid phase extraction (SPE) and ion exchange chromatography (IEX). The separation of shock lymph by both methodologies yielded two fractions having major detectable toxicity to endothelial cells, whereas no toxicity was detected from sham lymph separations by either method. Subsequent analysis of each SPE toxic fraction by gel electrophoresis and mass spectrometry suggests the toxicity is associated with a modified form of rat serum albumin (mod-RSA) and multiple lipid-based factors. Therefore, we have been able to demonstrate by two different separation techniques that shock lymph contains two or more factors that may account for the toxicity to endothelial cells. Further investigations are needed to determine the type of RSA modification and the identity of the lipid factors and their role in MODS.

Adenosine A2A receptor activation reduces lung injury in trauma/hemorrhagic shock.

Objective:Hemorrhagic shock and resuscitation trigger a global ischemia/reperfusion phenomenon, in which various inflammatory processes critically contribute to the ensuing tissue damage. Adenosine is an endogenous nucleoside that is released during shock. Activation of adenosine A2A receptors can broadly inactivate inflammatory cascades. The current study was designed to evaluate the effect of A2A receptor activation on organ injury and inflammation in the setting of global ischemia/reperfusion elicited by trauma/hemorrhagic shock and resuscitation. Design:Prospective animal study with concurrent control. Setting:Small animal laboratory. Subjects:Adult male Sprague-Dawley rats. Interventions:The rats were subjected to a laparotomy (trauma) and 90 mins of hemorrhagic shock or trauma/sham shock. The selective A2A receptor agonist CGS-21680 (2-p-(2-carboxyethyl) phenethylamino-5′-N-ethyl-carboxamidoadenosine; 0.5 mg/kg) or its vehicle was injected 30 mins before shock or immediately after resuscitation. At 3 hrs following resuscitation, animals were killed and tissue was harvested for analysis. Lung permeability and pulmonary myeloperoxidase levels were used to quantitate lung injury. Intestinal injury was determined by histologic analysis of terminal ileum. Red blood cell deformability was measured by a laser-assisted ektacytometer. In this assay, a decrease in the elongation index is a marker of decreased red blood cell deformability. Measurements and Main Results:Pretreatment with CGS-21680 protected the lung but not the gut against shock-induced injury and prevented the shock-induced decrease in red blood cell deformability. Posttreatment with CGS-21680 ameliorated shock-induced lung injury but failed to prevent gut injury and preserve red blood cell deformability. Conclusion:A2A receptor agonists may represent a novel therapeutic approach in preventing organ injury following trauma/hemorrhagic shock.

Effect of trauma-hemorrhagic shock on red blood cell deformability and shape.

Previous work in our laboratory has demonstrated a decrease in red blood cell (RBC) deformability in sepsis. This has not been studied following hemorrhagic shock. We tested the hypotheses that hemorrhagic shock, associated with soft tissue trauma, leads to decreased RBC deformability and that this is related to alterations in the resting shape of the RBC. Elongation index (EI), a measure of RBC deformability, was determined over a range of shear stresses from 0.3 to 30 Pa in 26 male rats before and at various times after 90 min of hemorrhagic shock. RBC resting shape was determined by scanning electron microscopy. The data demonstrate that EI decreased significantly at the end of shock (before resuscitation), and remained below normal throughout the 6-h postshock period. Eight of the 26 animals decompensated during shock, requiring return of a portion of the shed blood to maintain a mean arterial pressure of 30–40 mmHg. Four of eight decompensated animals died before the end of the study period, compared with none of the compensated rats. The decompensated rats had significantly lower EI at 0.3 Pa by the end of the shock period (0.050 ± 0.009) than the compensated shock group (0.058 ± 0.006;P < 0.05). RBC shape alterations were first demonstrated at the end of the shock period and persisted throughout the 6-h postshock resuscitation period. These data indicate that trauma and hemorrhagic shock cause RBC shape alterations and a significant decrease in RBC deformability, which becomes manifested during the shock period and persists for at least 6 h postshock. Additionally, a direct relationship appears to exist between the magnitude of the physiologic insult and the degree of RBC damage.

Trauma-hemorrhagic shock-induced red blood cell damage leads to decreased microcirculatory blood flow*

Objective:To test the hypothesis that trauma-hemorrhagic shock (T/HS)-induced changes in red blood cells (RBC) contribute to the reduction of blood flow in distant organs. Design:Laboratory study. Setting:Academic medical center laboratory. Subjects:Specific pathogen-free male Sprague-Dawley rats weighing between 250 and 350 g. Interventions:Rats were transfused with trauma-sham shock (T/SS), or T/HS whole blood, or RBC-depleted blood (blood with the RBC removed and consisting of white blood cells and plasma). Measurements and Main Results:Cardiac output and organ blood flow were measured by the radioactive microsphere technique. RBC tissue trapping, deformability, and RBC aggregation and adhesion were studied. Measurements of RBC adenosine triphosphate (ATP) and plasma fibrinogen were performed. Exchange transfusion with T/SS blood did not alter cardiac output or organ blood flow. However, cardiac output and blood flow in several organs were decreased when T/HS whole blood was used and RBCs were trapped in the organs that evidenced decreased blood flow. T/HS also increased RBC aggregation and adhesion, and decreased deformability. The ability of T/HS exchange transfusion to decrease microcirculatory blood flow did not appear to be due to plasma factors or non-RBC elements (i.e., white blood cell), because organ blood flow was not reduced after exchange transfusion with T/HS RBC-depleted blood. Likewise, neither decreased RBC ATP nor increased plasma fibrinogen explained the T/HS-induced changes that were observed. There was no change in fibrinogen levels during or after shock. Although there was a transient decrease in T/HS erythrocyte ATP levels during the early shock period, in contrast to RBC function, the ATP levels had returned to normal with resuscitation. Conclusions:T/HS induces significant changes in RBC functions and the injection of T/HS, but not T/SS, RBC leads to decreased organ blood flow. These findings confirm the hypothesis that T/HS-induced RBC alterations will directly cause organ hypoperfusion and suggest that T/HS-induced RBC damage contributes to this process. Thus, T/HS-induced changes in RBC function may contribute to the development of shock-induced multiple organ failure.

Albumin protects against gut-induced lung injury in vitro and in vivo.

Objective:Since albumin has the ability to detoxify, we assessed whether low-dose albumin could protect against trauma/hemorrhagic shock (T/HS)-induced endothelial cell, lung, gut, and red blood cell (RBC) injury in vivo and endothelial cell injury in vitro. Summary Background Data:T/HS cause ischemic insult to the gut, resulting in the release of biologically active factors into the mesenteric lymph, which then cause injury to multiple distant organs. Methods:In vitro experiments tested the ability of albumin to reduce the cytotoxicity of mesenteric lymph from male rats subjected to T/HS (laparotomy + MAP 30 mm Hg for 90 minutes) for human umbilical vein endothelial cell (HUVEC). In subsequent in vivo experiments, the ability of albumin given as part of the resuscitation regimen to protect against T/HS-induced injury was tested by comparing the magnitude of injury in T/HS rats receiving human albumin (shed blood + 0.12, 0.24, or 0.36 g/kg) or lactated Ringers solution (shed blood + 2 × volume of shed blood as LR) with that observed in rats subjected to trauma/sham shock. Rats were killed after a 3-hour recovery period and had lung permeability evaluated by bronchoalveolar lavage and myeloperoxidase assays, intestinal microvillous injury by histology, and RBC deformability using ektacytometry. Results:Both bovine and human albumin prevented T/HS lymph-induced HUVEC cytotoxicity in vitro, even when added 30 minutes after the lymph (viability 15 ± 4% to 88 ± 3%, P < 0.01). In vivo RBC deformability was better preserved by blood plus albumin than blood plus lactated Ringers solution (P < 0.01). Likewise, albumin administration reduced T/HS-induced lung permeability and neutrophil sequestration in a dose-dependent fashion, with 0.36 g/kg of albumin effecting total lung protection (P < 0.01). In contrast, albumin treatment did not prevent T/HS-induced gut injury. Conclusions:Low-dose albumin protects against gut lymph-induced lung, HUVEC, and RBC injury by neutralizing T/HS lymph toxicity.

Mesenteric lymph duct ligation prevents shock-induced RBC deformability and shape changes

OBJECTIVES The exact mechanisms that lead to RBC deformability and shape changes after trauma/hemorrhagic shock remain unknown. We hypothesize that RBC injury is caused in part by gut injury and is mediated by gut-derived factors carried in the intestinal lymph. MATERIALS AND METHODS RBC deformability was measured by a laser-assisted ektacytometer before and after trauma/hemorrhagic shock (T/HS) in 6 rats whose mesenteric lymph duct had been ligated and in 10 rats subjected to T/HS without duct ligation. In this assay a decrease in the elongation index is a marker of decreased RBC deformability. RBC shape was examined by scanning electron microscopy. RESULTS In the T/HS rats, the RBC elongation index decreased after T/HS from a preshock value of 0.064 +/- 0.011 to 0.052 +/- 0.009 (P < 0.01) and remained low (0.049 +/- 0.010) even at 3 h after resuscitation. In contrast, the elongation index did not decrease after T/HS in the lymph duct-ligated rats (0.062 +/- 0.004 vs. 0.056 +/- 0.005, P = NS). Likewise, the T/HS rats, but not the duct-ligated T/HS rats, had a significant increase in the percentage of abnormally shaped RBCs when studied by electron microscopy. CONCLUSIONS Interruption of lymph flow from the gut into the bloodstream by lymph duct ligation prevents T/HS-induced RBC damage. Because decreased RBC deformability contributes to impaired perfusion of the microcirculation, preservation of RBC deformability may decrease the incidence of T/HS-induced organ dysfunction.

Pancreatic Duct Ligation Reduces Lung Injury Following Trauma and Hemorrhagic Shock

Objective:To determine whether pancreatic digestive enzymes released into the ischemic gut during an episode of T/HS are involved in the generation of distant organ injury. This hypothesis was tested by examining the effect of PDL on T/HS-induced intestinal injury, lung injury, and RBC deformability. Summary Background Data:The effect of pancreatic duct ligation (PDL) on distant organ injury following trauma/hemorrhagic shock (T/HS) was examined. PDL before T/HS decreases lung and red blood cell (RBC) injury and exerts a limited protective effect on the gut. Pancreatic proteases in the ischemic gut appear to be involved in gut-induced lung and RBC injury. Based on recent work, it appears that proinflammatory and/or toxic factors, which are generated by the ischemic intestine, play an important role in the pathogenesis of multiple organ failure. The process by which these toxic factors are generated remains unknown. Previous experimental work has clearly documented that intraluminal inhibition of pancreatic proteases decreases the degree of T/HS-induced lung injury and neutrophil activation. One possible explanation for this observation is that the toxic factors present in intestinal lymph are byproducts of interactions between pancreatic proteases and the ischemic gut. Methods:Male Sprague-Dawley rats were subjected to a laparotomy (trauma) and 90 minutes of sham (T/SS) or T/HS with or without PDL. At 3 and 24 hours following resuscitation, animals were killed and samples of gut, lung, and blood were collected for analysis. Lung permeability, pulmonary myeloperoxidase levels, and bronchoalveolar fluid protein content were used to quantitate lung injury. Intestinal injury was determined by histologic analysis of terminal ileum (% villi injured). To assess RBC injury, RBC deformability was measured, as the RBC elongation index (RBC-EI), using a LORCA device. Results:At 3 and 24 hours following resuscitation, PDL prevented shock-induced increases in lung permeability to both Evans blue dye and protein in addition to preventing an increase in pulmonary myeloperoxidase levels. T/HS-induced impairments in RBC deformability were significantly reduced at both time points in the PDL + T/HS group, but deformability did not return to T/SS levels. PDL did reduce the magnitude of ileal injury at 3 hours after T/HS, but the protective effect was lost at 24 hours after T/HS. Conclusions:PDL prior to T/HS decreases lung injury and improves RBC deformability but exerts a limited protective effect on the gut. Thus, the presence of pancreatic digestive enzymes in the ischemic gut appears to be involved in gut-induced lung and RBC injury.

Resistance of the female, as opposed to the male, intestine to I/R-mediated injury is associated with increased resistance to gut-induced distant organ injury.

We tested the hypothesis that the female intestine is more resistant to gut I/R injury than the male intestine by comparing the effects of the isolated pure gut I/R superior mesenteric artery occlusion (SMAO) model on gut morphology and whether SMAO-induced distant organ injury (lung, bone marrow [BM], neutrophils, and red blood cells [RBCs]) would differ between male and proestrus female rats. At 6 or 24 h after SMAO or sham SMAO, gut injury, lung permeability, pulmonary neutrophil sequestration, RBC deformability, and BM RBC and white blood cell progenitor growth were measured, as was the ability of the plasma from these rats to activate naive rat neutrophils. At both 6 and 24 h after SMAO, the female rats had significantly less intestinal injury and reduced gut-induced lung injury, BM suppression, RBC dysfunction, and neutrophil activation than male rats subjected to SMAO. These results indicate that the resistance of proestrus female rats to gut injury and gut-induced distant organ injury is greater than that observed in male rats.

Microscopic and Macroscopic Evaluation of Emboli Captured during Angioplasty and Stent Procedures in Extracranial Vertebral and Internal Carotid Arteries

Purpose: To compare the quantities of emboli dislodged during percutaneous transluminal angioplasty/stenting in the vertebral artery (VA) with those released during stent placement in the internal carotid artery (ICA). Methods: Macroscopic images of distal protection devices (DPD) used during 30 stent procedures in 16 ICAs (11 men; mean age 64.6±10.6 years) and 14 VAs (9 men; mean age 67.1±9.8 years) were reviewed. The amount of captured embolic debris was calculated and expressed as a proportion to the size of the filter. Histological examinations were performed to characterize the material trapped in the filters. Results: Relative to the size of the filter, the proportion of captured debris ranged from 0.1% to ∼22% in the ostial VA filters and from 0.1% to ∼21% in the filters used in the ICA procedures (p=NS). Plaque fragments with or without thrombus were discovered in the histological examinations of captured material. There were no significant differences in the characteristics of the debris between the 2 vascular regions, nor did sex, race, or plaque morphology correlate significantly with the proportion of captured debris. However, the severity of stenosis was significantly (p<0.029) greater in the ICA (73%±0.11%) than the VA (63%±0.09%) territory. Conclusion: The study suggests that the frequency and amount of captured emboli during stent procedures in ICA and ostial VAs are comparable. Therefore, the use of a DPD for stent placement in the vertebral artery may be advisable.

Amiloride combined with small-volume resuscitation with hypertonic saline is superior in ameliorating trauma-hemorrhagic shock-induced lung injury in rats to the administration of either agent alone

Objective:Recognition of the limitations of standard crystalloid resuscitation has led to exploration for alternative resuscitation strategies that might better prevent the development of trauma-hemorrhage-induced organ dysfunction and systemic inflammation. Thus, the goal of this study was to compare the effects of two resuscitation strategies alone and in combination with that of standard resuscitation with Ringer’s lactate. These two strategies were intravenous injection of amiloride, an inhibitor of Na+/H+ exchange and epithelial Na+ channels, and resuscitation with hypertonic saline. Design:Prospective animal study with concurrent control. Setting:Small animal laboratory. Subjects:Adult male Sprague-Dawley rats. Interventions:Rats injected with amiloride or its vehicle were subjected to trauma-hemorrhagic shock (T/HS) or trauma sham-shock (T/SS) and resuscitated with Ringer’s lactate or hypertonic saline. The T/HS model consisted of a laparotomy plus 90 mins of shock (mean arterial pressure 30 mm Hg). Three hours after the end of the shock or sham-shock period, lung permeability, lung histology, pulmonary neutrophil sequestration, neutrophil CD11b expression, gut injury, and red blood cell rigidification were assessed. Measurements and Main Results:Both amiloride and hypertonic saline reduced T/HS-induced pulmonary permeability and neutrophil sequestration, and coadministration of these two agents was more efficacious than administration of the individual agents. In contrast, whereas gut injury was attenuated by both amiloride and hypertonic saline, combined administration of amiloride and hypertonic saline failed to further protect the gut. Additionally, hypertonic saline reduced both neutrophil CD11b expression and red blood cell rigidification, whereas amiloride was without effect. Conclusions:Combined administration of amiloride and small-volume resuscitation with hypertonic saline may be a strategy worthy of further evaluation in the therapy of shock-induced distant organ injury.