Glutathione deficiency increases organ dysfunction after hemorrhagic shock.

BACKGROUND. Reactive oxygen metabolites contribute to tissue destruction in a wide variety of diseases. Glutathione, a potent endogenous antioxidant, neutralizes the destructive potential of free radicals, but this tripeptide may be depleted during illness. We hypothesized that glutathione deficiency would amplify organ dysfunction after shock in rats. METHODS. Rats received either diethyl maleate to deplete tissue glutathione or a control solution intraperitoneally. The animals were subsequently bled to and maintained at a mean arterial pressure of 40 mm Hg for 30 minutes and then fully resuscitated. Sham animals underwent blood pressure monitoring only. Tissue glutathione, liver and renal function tests, organ bacterial content, and mortality rates were determined 4 and 24 hours after shock. RESULTS. Normal rats subjected to shock and sham animals had similar laboratory chemistry results, organ culture results, and mortality rates. However, glutathione-depleted animals subjected to shock had elevated liver and renal function tests, increased organ bacteria, and a dramatic increase in mortality rates compared with control shock and sham animals. CONCLUSIONS. We conclude that glutathione deficiency predisposes animals to organ failure and death after an otherwise nonlethal period of hypotension. Because glutathione deficiency is associated with severe injury and sepsis, treatment strategies that maintain glutathione stores may decrease the incidence of multisystem organ failure.     

Administration of progesterone after trauma and hemorrhagic shock prevents hepatocellular injury

HYPOTHESIS: Administration of a single dose of progesterone following trauma and hemorrhage in progesterone-deficient rats would ameliorate the inflammatory response and hepatocellular damage. SETTING: A university laboratory. INTERVENTIONS: Ovariectomized female Sprague-Dawley rats (250-350 g; Charles River Laboratories, Wilmington, Mass) underwent a 5-cm midline laparotomy (ie, induction of soft tissue trauma), were bled to a mean arterial blood pressure of 35 mm Hg for about 90 minutes, and then were resuscitated using Ringer lactate solution. Progesterone (25 mg/kg of body weight) or vehicle was administered subcutaneously at the end of resuscitation. In additional animals, Kupffer cells were isolated following trauma, hemorrhage, and resuscitation and treated in vitro with progesterone, lipopolysaccharide, or both. MAIN OUTCOME MEASURES: Six hours following resuscitation, plasma tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) levels and liver myeloperoxidase activity were determined. Hepatocellular function (maximum velocity of indocyanine green clearance [Vmax] and the efficiency of the active transport or Michaelis-Menten constant [Km]) and plasma levels of transaminases were measured 20 hours after resuscitation. Kupffer cell IL-6 and TNF-alpha production were assessed. RESULTS: Plasma levels of TNF-alpha, IL-6, aspartate aminotransferase, and alanine aminotransferase, as well as hepatic myeloperoxidase activity were increased, whereas indocyanine green clearance was depressed in vehicle-treated rats following trauma-hemorrhage. Animals treated with progesterone showed significantly reduced levels of the TNF-alpha, IL-6, and transaminases as well as reduced myeloperoxidase activity in the liver. Progesterone-treated animals showed increased Vmax and Kmax values for indocyanine green. In vitro treatment of Kupffer cells with progesterone decreased TNF-alpha production but did not affect the production of IL-6. CONCLUSION: Progesterone administration following trauma-hemorrhage ameliorates the proinflammatory response and, subsequently, the hepatocellular injury via direct action on immunocompetent cells.     

Reperfusion inhibits elevated splanchnic prostanoid production after hemorrhagic shock.

The effect of reperfusion following hemorrhagic shock on splanchnic prostanoid release was studied. Anesthetized male rats were bled to a mean arterial blood pressure of 30 mmHg for 30 minutes and either killed or treated with shed blood for 60 minutes and then killed. The superior mesenteric arterial bed was cannulated and perfused in vitro with oxygenated Krebs. Collected venous effluent (up to 180 minutes) was analyzed for 6-keto-PGF1 alpha (PGI2 metabolite), PGE2, PGF2 alpha, and thromboxane B2 by radioimmunoassay in shock, shock plus reperfusion, and sham groups. The major prostanoid released was 6-keto-PGF1 alpha and was three times higher in the shock group compared to the sham group (p less than 0.05). Reperfusion of shed blood abolished the increase in 6-keto-PGF1 alpha found in the shock group (p less than 0.05). These data show that the attempt of the rat splanchnic bed to compensate for hemorrhagic shock by increasing release of PGI2 (potent vasodilator) was abolished during reperfusion of blood.     

Antioxidative resuscitation solution prevents leukocyte adhesion in the liver after hemorrhagic shock.

BACKGROUND: The generation of iron-dependent toxic oxygen radicals during the initial resuscitation from hemorrhagic shock was shown to be a relevant factor for the initiation of the inflammatory cascade. Therefore, this experimental study was designed to evaluate the effects of a deferoxamine-conjugated hydroxyethyl-starch solution (HES-DFO) on oxygen radical induced injury and microcirculatory alterations in the rat liver compared with resuscitation with regular hydroxyethyl-starch, lactated Ringer's solution (RL), or a gelatin-based solution. METHODS: After hemorrhage and random assignment to 1 hour of blood-free resuscitation with the aforementioned solutions, hepatic microcirculation and leukocyte adhesion characteristics were assessed by intravital fluorescence microscopy in anesthetized rats. Oxygen radical activity was estimated by determination of glutathione levels in liver homogenate and determination of thiobarbituric acid-reactive substances in plasma as markers of lipid peroxidation. RESULTS: Resuscitation by HES-DFO resulted in restoration of hemodynamic parameters compared with gelatin-based solution and HES. The hepatic microcirculation was severely altered 1 hour after resuscitation from shock in all groups indicated by sinusoidal narrowing and reduced sinusoidal blood flow. HES-DFO, however, attenuated leukocyte adhesion and improved velocity index in sinusoids as well as sinusoidal perfusion. The shock-associated generation of oxygen radicals during resuscitation was prevented by HES-DFO as indicated by restored glutathione and reduced thiobarbituric acid-reactive substances. CONCLUSION: The results suggest that HES-DFO effectively reduces oxygen radical formation during the initial resuscitation period, thus, attenuating pathologically enhanced leukocyte adhesion and improving hepatic microcirculation.     

Moderate hypothermia prevents brain stem oxidative stress injury after hemorrhagic shock

BACKGROUND: The purpose of this study was to investigate the effects of temperature on oxidative stress in brain stem tissue induced by hemorrhagic shock. We researched the hemorrhagic oxidative stress at various core temperatures using reduced glutathione (GSH) levels and thiobarbituric acid-reactive substances (TBARS) as markers of lipid peroxidation in brain stem homogenate. METHODS: Forty rats were divided into four groups, of which one constituted the nonbleeding normothermia control group. In all of the three study groups, 40% of estimated blood volume was removed while they were being held at normothermia, mild hypothermia (32 degrees C), or moderate hypothermia (28 degrees C). Parameters including mean arterial pressure, rectal temperature, and heart and breathing rates were monitored and recorded during the procedures. After an hour at shock state, tissue samples were removed by craniectomy. RESULTS: The tissue levels of TBARS increased significantly in normothermic and mild hypothermic hemorrhagic shock groups (10.74 nmol/g and 8.26 nmol/g) as compared with the control group (3.50 nmol/g) (p < 0.001). However, the tissue TBARS level in the moderate hypothermia group was only minimally increased (4.53 nmol/g). GSH showed a slight decrease in normothermic and mild hypothermic bleeding rats, and were unchanged in the moderate hypothermic rats. CONCLUSION: Moderate systemic hypothermia (28 degrees C) appears to protect brain stem tissue from oxidative stress during severe hemorrhagic shock in rats, as indicated by insignificant change in tissue TBARS and GSH concentrations. These results suggest antioxidant protective effects of moderate systemic hypothermia in metabolically active brain stem tissue during hemorrhagic shock. Similar effects in humans remain to be studied.     

Myocardial oxygen delivery after experimental hemorrhagic shock.

The two components of myocardial oxygen delivery, coronary blood flow to capillaries and diffusion from capillaries to mitochondria, were studied in six dogs, (1) prior to shock, (2) after three hours of hemorrhage shock at a mean systemic arterial pressure of 40 torr, (3) after reinfusion of shed blood, and (4) during the irreversible late posttransfusion stage. There was a maldistribution of left ventricular coronary flow during late shock consistent with subendocardial ischemia. Cardiac performance was significantly impaired after resuscitation and all dogs became irreversible. Total and regional left ventricular coronary blood flow and myocardial oxygen delivery to capillaries were significantly greater than preshock values in (3) but not different from preshock values in (4). However, the myocardial oxygen diffusion area to distance ratio was significantly lower than preshock values in (3), and slightly lower in (4). These data suggest that myocardial oxygen diffusion may be impaired in the early post transfusion period, (3). Accordingly, the probable etiology of left ventricular dysfunction and possibly irreversibility after resuscitation from hemorrhagic shock is subendocardial ischemia during shock with either post-resuscitation impairment of myocardial oxygen diffusion, or in cellular oxygen utilization, or both.     

Hyperoxic condition prevents bacterial translocation and elevation of plasma microorganism components during hemorrhagic shock.

To evaluate the influence of hyperoxic conditions on bacterial translocation (BT) and microorganism components during hemorrhagic shock, rats were divided into a group breathing 100% oxygen and a group breathing room air. The groups were then subjected to hemorrhagic shock. Systemic blood and mesenteric lymph nodes were cultured for BT, and systemic plasma concentrations of microorganism components were measured by the silkworm larvae plasma (SLP) test and the endotoxin test. Hyperoxic conditions prevented both BT and plasma SLP-reactive substance (peptidoglycan and beta-glucan) elevation during hemorrhagic shock. Our findings suggest that hyperoxic treatment might improve host conditions during hemorrhagic shock.     

Hypertonic resuscitation of hemorrhagic shock prevents alveolar macrophage activation by preventing systemic oxidative stress due to gut ischemia/reperfusion

BACKGROUND: The gut is a target organ of shock/resuscitation (S/R); however, it also contributes to distant inflammation through the generation of oxidants. S/R with antioxidants such as N-acetylcysteine (NAC) prevents lipopolysaccharide (LPS)-induced cytokine production and NF-kappaB activation in rat alveolar macrophages. Therefore, we hypothesized that hypertonic saline (HTS) might exerts its protective effect by preventing gut ischemia/reperfusion injury, thus decreasing oxidative stress and distant priming in alveolar macrophages. METHODS: A two-hit rat model of shock resuscitation was used. Plasma levels of 8-iso-prostaglandin, a marker of lipid peroxidation, was quantified by eicosanoid immunoassay with acetylcholinesterase kit. Gut histology with hematoxylin and eosin staining was performed 1 to 6 hours after resuscitation. Alternatively, alveolar macrophages from bronchoalveolar lavage (BAL) at end resuscitation were incubated in vitro with LPS (0.01 mug/mL), and NF-kappaB translocation was observed by immunofluorescent staining with anti-p65 antibody. RESULTS: HTS resuscitation prevented leukosequestration in the alveolar space, and it abrogated the progressive rise in blood 8-iso-prostaglandin production observed with Ringer's lactate (RL) resuscitation. Inhibition of oxidant stress with NAC corresponded with the ability of HTS to prevent S/R-induced edema, villus flattening, and mucosal sloughing in the mid-ileum. LPS-induced NF-kappaB translocation in alveolar macrophages after RL was 42% compared to 20% after HTS. Similar attenuation was observed with NAC resuscitation (16%). CONCLUSIONS: HTS resuscitation prevents systemic oxidative stress by reducing gut ischemia/reperfusion injury and consequently attenuates distant alveolar macrophage priming, thereby reducing LPS-induced NF-kappaB nuclear translocation in alveolar macrophages and organ injury. This represents a novel mechanism whereby HTS exerts its immunomodulatory effects.     

More is better. Antibiotic management after hemorrhagic shock.

Previous reports suggest that standard antibiotic prophylaxis is ineffective in reducing the incidence of wound infection after hemorrhagic shock. This study investigated the use of larger and longer doses of antibiotic in a model of staphylococcal infection after hemorrhagic shock. Sprague-Dawley rats resuscitated from hemorrhagic shock were injected with either 10(6), 10(8) or 10(10) Staphylococcus aureus subcutaneously. Five treatments were investigated: 1) control (no antibiotic), 2) short-course cefazolin (CEF) (SHORT), 30 mg/kg intraperitoneal (IP), 30 minutes before and 4 hours after inoculation, 3) long-course CEF (LONG), 30 mg/kg IP, 30 minutes before and 4 hours after inoculation, and thereafter, every 8 hours for 3 days, 4) mega-CEF (MEGA) 200 mg/kg IP, 30 minutes before and 4 hours after inoculation, and 5) mega-long CEF (MEGA-LONG), 200 mg/kg IP, 30 minutes before and 4 hours after inoculation, and thereafter, every 8 hours for 3 days. Abscess number, weight, and diameter were measured on Day 7. At the 10(6) inoculum, SHORT was effective in both shocked and unshocked animals. In the 10(10) group, all antibiotic regimens decreased the 100% mortality that followed shock without treatment, but they had little effect on abscess formation. In unshocked rats at the 10(8) inoculum, SHORT was effective in reducing abscess number, diameter, and weight (all p less than 0.05 vs. control). After hemorrhagic shock, SHORT did not decrease abscess frequency, but it did diminish abscess diameter. LONG significantly decreased abscess diameter and abscess weight (both p less than 0.05). After shock, both MEGA and MEGA-LONG reduced abscess number (p less than 0.05 vs. control) and MEGA-LONG was superior to all other regimens at the 10(8) inoculum. These experimental data show that increasing both the dose and duration of antibiotic administration is more effective than standard short-course antibiotic prophylaxis in preventing experimental infection after hemorrhagic shock.     

Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock.

OBJECTIVE: To determine whether gut-derived factors leading to organ injury and increased endothelial cell permeability would be present in the mesenteric lymph at higher levels than in the portal blood of rats subjected to hemorrhagic shock. This hypothesis was tested by examining the effect of portal blood plasma and mesenteric lymph on endothelial cell monolayers and the interruption of mesenteric lymph flow on shock-induced lung injury. SUMMARY BACKGROUND DATA: The absence of detectable bacteremia or endotoxemia in the portal blood of trauma victims casts doubt on the role of the gut in the generation of multiple organ failure. Nevertheless, previous experimental work has clearly documented the connection between shock and gut injury as well as the concept of gut-induced sepsis and distant organ failure. One explanation for this apparent paradox would be that gut-derived inflammatory factors are reaching the lung and systemic circulation via the gut lymphatics rather than the portal circulation. METHODS: Human umbilical vein endothelial cell monolayers, grown in two-compartment systems, were exposed to media, sham-shock, or postshock portal blood plasma or lymph, and permeability to rhodamine (10K) was measured. Sprague-Dawley rats were subjected to 90 minutes of sham or actual shock and shock plus lymphatic division (before and after shock). Lung permeability, pulmonary myeloperoxidase levels, alveolar apoptosis, and bronchoalveolar fluid protein content were used to quantitate lung injury. RESULTS: Postshock lymph increased endothelial cell monolayer permeability but not postshock plasma, sham-shock lymph/plasma, or medium. Lymphatic division before hemorrhagic shock prevented shock-induced increases in lung permeability to Evans blue dye and alveolar apoptosis and reduced pulmonary MPO levels. In contrast, division of the mesenteric lymphatics at the end of the shock period but before reperfusion ameliorated but failed to prevent increased lung permeability, alveolar apoptosis, and MPO accumulation. CONCLUSIONS: Gut barrier failure after hemorrhagic shock may be involved in the pathogenesis of shock-induced distant organ injury via gut-derived factors carried in the mesenteric lymph rather than the portal circulation.     

Gallbladder and gastrointestinal motility after hemorrhagic shock

The alterations in fasting gallbladder and gastrointestinal motility during hemorrhagic shock were investigated. Eight opossums implanted with a gallbladder cannula, gastrointestinal bipolar electrodes, and a carotid catheter were subjected to hemorrhagic shock of 30 mm Hg for 60 minutes by the removal of arterial blood. Shed blood was reinfused after the shock period. Fasting gallbladder volume and gastrointestinal electrical activity were studied before, immediately after, and 24 hours after hemorrhagic shock. Control measurements demonstrated a slow-wave frequency maximal in the duodenum (18.1 +/- 1.1 waves/min), with a plateau in the proximal third of the small bowel, decreasing thereafter. The migrating motor complex (MMC) had a duration of 118 +/- 28 minutes. The average volume of the gallbladder before shock was 5.4 +/- 1.5 ml. Gallbladder volume fluctuated with the MMC, being maximal during phase I and minimal in phase III. The volume of blood removed to reduce mean arterial pressure to 30 mm Hg was 45 +/- 5 ml/kg. Immediately after the shock and blood reinfusion, slow-wave frequency decreased by 40% in the antrum and 25% in the small bowel. The MMC was of shorter duration (91 +/- 22 minutes; p less than 0.05), and gallbladder volume increased to 7.0 +/- 1.7 ml (p less than 0.05). Fluctuations in gallbladder volume during the MMC were absent. Twenty-four hours after shock, slow-wave frequency, MMC, and gallbladder volume had returned to normal and were not different from control measurements. Ischemic damage to the gastrointestinal tract is postulated as the cause of gallbladder dysfunction and altered intestinal motility after hemorrhagic shock.     

Splanchnic prostanoid production: effect of hemorrhagic shock

The effect of hemorrhagic shock on rat splanchnic prostanoid synthesis was examined in vitro. Male rats were bled to a mean pressure of 30 mm Hg for 30 min. At that time a midline laparotomy was performed and the superior mesenteric artery (SMA) was cannulated and removed in continuity with its end organ (SV + SI). The SV + SI was perfused at 3 ml/min with oxygenated Krebs-Henseleit buffer at 37 degrees C. Venous effluent (3 ml) was collected at 15, 30, 60, 90, 120, 150, and 180 min and frozen for analysis of basal prostanoid release. Levels of 6-keto-PGF1 alpha (6-keto), PGE2, thromboxane B2 (TxB2), and PGF2 alpha were analyzed by radioimmunoassay and compared in the shock (SK) and sham-operated control (SM) animals. SK 6-keto and TxB2 increased significantly at 15 min of perfusion and then decreased to a lower baseline from 60 through 120 min of perfusion. The major prostanoid released at all time periods was 6-keto (2 to 20 times higher than other prostanoids). One-way ANOVA (pooled times) showed that 6-keto significantly increased in the SK group compared to the SM group (P less than 0.02). These data suggest that the splanchnic bed attempts to compensate for hemorrhagic shock by the marked release of the endogenous vasodilator prostacyclin. Release of the other prostanoids (including TxB2) from the splanchnic bed was modest in our model.     

Neutrophil regulation of splanchnic blood flow after hemorrhagic shock.

OBJECTIVE: This study examines the hypothesis that neutrophils impair splanchnic blood flow during resuscitation from hemorrhage by inhibiting the release of the compensatory vasodilator PGI2 from the bowel. SUMMARY BACKGROUND DATA: Resuscitation from hemorrhagic shock is associated with neutrophil infiltration into the intestine, reduced splanchnic perfusion, and reduced release of PGI2 from the intestine. METHODS: Sprague-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 PGI2) release were measured. RESULTS: Superior 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). CONCLUSION: These data are compatible with the hypothesis that neutrophils may influence splanchnic perfusion after SK + R by inhibiting splanchnic PGI2 release.     

Occurrence of bacteremia during and after hemorrhagic shock

In recent research, hemorrhagic shock and septic shock have been studied as two separate entities. We have developed a treated model of hemorrhagic shock in which unrestrained and unanesthetized rats are bled to a mean arterial pressure of 30 torr until 80% of the maximum shed volume must be returned. Rats are maintained preshock and treated post shock with a 20% glucose-electrolyte solution. Survival of these animals is 62% at 24 hours post shock and all animals are dead at 72 hours post shock. Blood cultures obtained during shock become positive at 2 hours into the shock period and are significant compared to controls at 3 to 5 hours of shock (p less than 0.0001). Blood cultures obtained after the period of shock are significantly positive at 24 and 48 hours post shock (p less than 0.05) compared to controls. Intrashock cultures are monomicrobial; the majority of post-shock cultures are polymicrobial. All cultured organisms are normal rat enteric flora. Histologic changes of renal failure are also demonstrated post shock. We suggest that bacterial invasion, possibly from the gut, plays a role in the sepsis seen in patients following severe hemorrhagic shock. Sepsis may precede rather than follow the immune incompetence which accompanies shock.     

Gender affects macrophage cytokine and prostaglandin E2 production and PGE2 receptor expression after trauma

BACKGROUND: Gender influences morbidity and mortality after injury. Hormonal differences are important; however, the role of prostaglandins as mediators in immune dysfunction relating to gender differences after trauma is unclear. We hypothesized that gender-dependent differences in PGE(2) receptor expression and signaling may be involved in immune-related differences. This study determined prostaglandin receptor subtype (EP1-EP4) expression following injury and determined whether gender differences influence EP receptor expression. MATERIALS AND METHODS: BALB/c male and female mice (estrus and pro-estrus) (n = 6 per group) were subjected to femur fracture and 40% hemorrhage (trauma) or sham injury (anesthesia). Seven days later, the splenic macrophages were harvested and stimulated with lipopolysaccharide (Escherichia coli serotype O55:B5). After 6 h mRNA samples were collected for EP receptor mRNA expression and at 24 h supernatants were collected for PGE(2), TNF-alpha, and IL-6 production. RESULTS: The expression of EP2-4 receptors was higher in female pro-estrus mice compared with male mice. EP1 receptor expression was higher in males than pro-estrus females. There was decreased expression of all four receptors after trauma in female estrus compared with control estrus mice. Macrophage PGE(2), TNF-alpha, and IL-6 production was significantly increased in injured female mice compared with female controls but there were no differences in injured male mice compared with male controls. PGE(2) and TNF-alpha production by traumatized male mice were significantly less than that produced by traumatized pro-estrus females. CONCLUSIONS: These data suggest gender-related differences in response to traumatic injury and that alterations in specific EP receptor subtypes may be involved in immune dysfunction after injury. Studies to evaluate targeted modulation of these receptor subtypes may provide further insights to gender-specific differences in the immune response after injury.     

Alteration of alpha-spectrin ubiquitination after hemorrhagic shock

Background

RBC deformability after trauma and hemorrhagic shock (T/HS) leads to the microcirculatory dysfunction and clinical manifestations of organ failure. However, the cellular mechanism of this phenomenon remains unknown. Spectrins are important for the shape and physical properties of erythrocytes, such as deformability and resistance to mechanical stress. Previous studies have shown that erythrocyte α-spectrin is ubiquitinated. Studies of sickled cells and aged erythrocytes, 2 conditions known to have decreased RBC deformability, have shown decreased α-spectrin ubiquitination, which may contribute to the inability of these cells to change shape. It was hypothesized that decreased α-spectrin ubiquitination could participate in the mechanism(s) whereby T/HS erythrocytes become less deformable.

Methods

The level of α-spectrin ubiquitination in erythrocytes isolated from T/HS rats was determined and compared with erythrocytes from control sham-shocked (T/SS) animals. After T/SS (n = 4) or T/HS (n = 7), α- and β-spectrin subunits were isolated using a low ionic-strength buffer at 37°C for 30 minutes. The relative amount of ubiquitinated α-spectrin was evaluated by Western blotting using a monoclonal antibody to ubiquitin.

Results

The relative level of α-spectrin ubiquitination (normalized to total α-spectrin in the same preparation) was found to be significantly decreased after T/HS (.319 ± .03) compared with T/SS control erythrocytes (.485 ± .06, P < .05). To evaluate the content and relative amounts of the other membrane proteins, the profiles of T/HS and T/SS erythrocytes were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This did not reveal any significant quantitative differences between T/SS and T/HS spectrin or other membrane proteins.

Conclusions

The finding of decreased α-spectrin ubiquitination after T/HS suggests that this mechanism could contribute to increased rigidity of the erythrocyte membrane.