Resuscitation affects microcirculatory polymorphonuclear leukocyte behavior after hemorrhagic shock: role of hypertonic saline and pentoxifylline

We have previously shown that lung injury following fluid resuscitation either with hypertonic saline (HS) or lactated Ringer's (LR) plus pentoxifylline (PTX) attenuated acute lung injury when compared with LR resuscitation. The objective of the present study is to determine whether our previous observations are accompanied by changes in polymorphonu-clear leukocyte (PMN) behavior. To study this, PMN-endothelial cell interactions, microcirculatory blood flow, lung histology, lung PMN infiltration (MPO, Myeloperoxidase), and lung intra-cellular adhesion molecule-1 (ICAM-1) expression were assessed in a controlled hemorrhagic shock model followed by LR, HS, and LR+PTX resuscitation in rodents. Rats (240-300 g) were bled to a mean arterial pressure (MAP) of 35 mm Hg for 1 hr and then randomized into three groups: HS (7.5% NaCl, 4 ml/kg); LR (3x shed blood); and LR+PTX (25 mg/kg). Additionally, total shed blood was reinfused. A sham group underwent no shock and no treatment. The internal spermatic fascia was exteriorized and the microcirculation was observed by closed-circuit TV coupled to a microscope, 2 and 6 hrs after treatment. The number of leukocytes sticking to the venular endothelium was determined 2 hrs after fluid resuscitation. Microcirculatory blood flow was measured by an optical Doppler velocimeter. Lung histology and lung MPO immunostaining were assessed at 6 hrs, and lung ICAM-1 expression was determined by immunostaining at 2 hrs following fluid resuscitation. Two hours after treatment, HS (1.4 +/- 0.4), LR+PTX (1.7 +/- 0.3), and sham (0.4 +/- 0.2) groups presented significant reductions in leukocyte adherence (cells/100 microm venule length), compared with the LR group (4.0 +/- 0.9, P < 0.05). No differences were observed 6 hrs after treatment on leukocyte adherence and microcirculatory blood flow. ICAM-1 expression was significantly higher in LR-treated animals compared with the HS, LR+PTX, and sham groups (P < 0.01). PMN infiltration and overall lung injury were significantly attenuated by HS and LR+PTX. These results support earlier studies that indicated the potential application of HS and PTX in shock therapy and the increase in PMN-endothelial cell interaction and lung injury after LR resuscitation.     

Hypertonic Saline Dextran Ameliorates Organ Damage in Beagle Hemorrhagic Shock

Objective

The goal of this study was to investigate the effect of hypertonic saline with 6% Dextran-70 (HSD) resuscitation on organ damage and the resuscitation efficiency of the combination of HSD and lactated ringers (LR) in a model of hemorrhage shock in dogs.

Methods

Beagles were bled to hold their mean arterial pressure (MAP) at 50±5 mmHg for 1 h. After hemorrhage, beagles were divided into three groups (n = 7) to receive pre-hospital resuscitation for 1 h (R1): HSD (4 ml/kg), LR (40 ml/kg), and HSD+LR (a combination of 4 ml/kg HSD and 40 ml/kg LR). Next, LR was transfused into all groups as in-hospital resuscitation (R2). After two hours of observation (R3), autologous blood was transfused. Hemodynamic responses and systemic oxygenation were measured at predetermined phases. Three days after resuscitation, the animals were sacrificed and tissues including kidney, lung, liver and intestinal were obtained for pathological analysis.

Results

Although the initial resuscitation with HSD was shown to be faster than LR with regard to an ascending MAP, the HSD group showed a similar hemodynamic performance compared to the LR group throughout the experiment. Compared with the LR group, the systemic oxygenation performance in the HSD group was similar but showed a lower venous-to-arterial CO₂ gradient (Pv-aCO₂) at R3 (p < 0.05). Additionally, the histology score of the kidneys, lungs and liver were significantly lower in the HSD group than in the LR group (p < 0.05). The HSD+LR group showed a superior hemodynamic response but higher extravascular lung water (EVLW) and lower arterial oxygen tension (PaO₂) than the other groups (p < 0.05). The HSD+LR group showed a marginally improved systemic oxygenation performance and lower histology score than other groups.

Conclusions

Resuscitation after hemorrhagic shock with a bolus of HSD showed a similar hemodynamic response compared with LR at ten times the volume of HSD, but HSD showed superior efficacy in organ protection. Our findings suggest that resuscitation with the combination of HSD and LR in the pre-hospital setting is an effective treatment.     

ATP-MgCl2 restores renal microcirculation following trauma and severe hemorrhage.

Although ATP-MgCl2 enhances the recovery of renal function after ischemia and reperfusion, it is not known whether this agent has any beneficial effects on renal microcirculation and function in a nonheparinized model of trauma and severe hemorrhage. To study this, a midline laparotomy was performed (i.e., trauma induced) and the rats were bled to and maintained at a mean arterial pressure of 40 mmHg (1 mmHg = 133.32 Pa) until 40% of the maximum shed blood volume was returned in the form of Ringer's lactate (RL) solution. Animals were then resuscitated with 4 times the volume of the shed blood in the form of RL. ATP-MgCl2, 50 mumol/kg body weight, or an equivalent volume of saline, was infused intravenously during and following resuscitation. Renal microcirculation was examined by using colloidal carbon infusion and laser Doppler flow-metry. Glomerular filtration rate (GFR) was assessed with [3H]inulin clearance and cardiac output (CO) was determined by dye dilution technique. The results indicate that the depressed renal microcirculation following hemorrhage and resuscitation was restored by ATP-MgCl2 treatment. GFR was significantly higher in ATP-MgCl2-treated than saline-treated rats. ATP-MgCl2 also increased urine output, restored the decreased CO, and prevented the occurrence of renal edema after hemorrhage and resuscitation. Thus, ATP-MgCl2 appears to be a useful adjunct to crystalloid resuscitation following trauma and severe hemorrhagic shock even in the absence of blood resuscitation.     

Comparison of transfusion with DCLHb or pRBCs for treatment of intraoperative anemia in sheep.

Isoflurane-anesthetized sheep were transfused with packed red blood cells (pRBCs) or diaspirin cross-linked hemoglobin (DCLHb) for treatment of intraoperative hemorrhage. A rapid 15-min hemorrhage with lactated Ringer (LR) infusion maintained filling pressure at baseline and reduced blood hemoglobin (Hb) to ~5 g/dl. Sheep received 2 g/kg Hb, DCLHb (n = 6), or pRBCs (n = 7); control group received LR alone (n = 6). After 2 h, anesthesia was discontinued; sheep were monitored in the animal intensive care unit for 48 h. DCLHb expanded blood volume more, but increased total blood Hb less, than pRBCs. Lower Hb and increased methemoglobin resulted in lower arterial oxygen content compared with the pRBCs. DCLHb caused pulmonary hypertension (from 13 to 30 mmHg) and elevated filling pressure (from 6 to 15 mmHg). Cardiac outputs (CO) were similar for all groups during anesthesia; however, during recovery CO increased only in the LR and packed pRBCs groups. DCLHb may limit the reflex ability to increase CO after volume expansion. Hemodynamic effects of DCLHb may be exaggerated when infused after large-volume LR.     

Downregulation of the Non-Integrin Laminin Receptor Reduces Cellular Viability by Inducing Apoptosis in Lung and Cervical Cancer Cells

The non-integrin laminin receptor, here designated the 37-kDa/67-kDa laminin receptor (LRP/LR), is involved in many physiologically relevant processes, as well as numerous pathological conditions. The overexpression of LRP/LR on various cancerous cell lines plays critical roles in tumour metastasis and angiogenesis. This study investigated whether LRP/LR is implicated in the maintenance of cellular viability in lung and cervical cancer cell lines. Here we show a significant reduction in cellular viability in the aforementioned cell lines as a result of the siRNA-mediated downregulation of LRP. This reduction in cellular viability is due to increased apoptotic processes, reflected by the loss of nuclear integrity and the significant increase in the activity of caspase-3. These results indicate that LRP/LR is involved in the maintenance of cellular viability in tumorigenic lung and cervix uteri cells through the blockage of apoptosis. Knockdown of LRP/LR by siRNA might represent an alternative therapeutic strategy for the treatment of lung and cervical cancer.     

17-DMAG diminishes hemorrhage-induced small intestine injury by elevating Bcl-2 protein and inhibiting iNOS pathway, TNF-α increase, and caspase-3 activation

Background

Hemorrhage increases inducible nitric oxide synthase (iNOS) and depletes ATP levels in various tissues. Previous studies have shown that geldanamycin, an inducer of heat shock protein 70kDa (HSP-70) and inhibitor of iNOS, limits both processes. Reduction in NO production limits lipid peroxidation, apoptosome formation, and caspase-3 activation, thereby increasing cellular survival and reducing the sequelae of hemorrhage. The poor solubility of geldanamycin in aqueous solutions, however, limits its effectiveness as a drug. 17-DMAG is a water-soluble analog of geldanamycin that might have greater therapeutic utility. This study investigated the effectiveness of 17-DMAG at reducing hemorrhagic injury in mouse small intestine.

Results

In mice, the hemorrhage-induced iNOS increase correlated with increases in Kruppel-like factor 6 (KLF6) and NF-kB and a decrease in KLF4. As a result, increases in NO production and lipid peroxidation occurred. Moreover, hemorrhage also resulted in decreased Bcl-2 and increased TNF-α, IL-6, and IL-10 concentrations, p53 protein, caspase-3 activation, and cellular ATP depletion. A shortening and widening of villi in the small intestine was also observed. Treatment with 17-DMAG significantly reduced the hemorrhage-induced increases in iNOS protein, jejunal alteration, and TNF-α and IL-10 concentrations, but 17-DMAG did not affect the hemorrhage-induced increases in p53 and IL-6 concentration. 17-DMAG treatment by itself upregulated HSP-70, Bcl-2, and p53.

Conclusion

Since 17-DMAG is water soluble, bioactive, and not toxic, 17-DMAG may prove useful as a prophylactic drug for hemorrhage.     

Fibrinogen availability and coagulation function after hemorrhage and resuscitation in pigs

Hemorrhagic coagulopathy (without neurological injuries) constitutes 40% of injury-related death in civilian hospitals and on the battlefield, and the underlying contributing mechanisms remain unclear. The purpose of this study is to investigate the effects of fibrinogen availability on coagulation function after hemorrhage in pigs. Sixteen crossbred commercial Yorkshire swine were randomized into the control group (group C) (n = 8) and hemorrhage group (group H) (n = 8). Hemorrhage was induced in group H by bleeding 35% of the estimated total blood volume, followed by resuscitation with lactated Ringer solution at three times the bled volume. Pigs in group C were not hemorrhaged or resuscitated. Blood samples were withdrawn at baseline, 15 min, 3 h, 6 h, and 24 h after hemorrhage and lactated Ringer (LR) resuscitation (H-LR). Coagulation was assessed by using thrombelastography. All baseline measurements were similar between groups C and H. Hemorrhage caused a decrease in mean arterial pressure and an increase in heart rate in group H, but LR resuscitation corrected these changes within 1 h. Compared to baseline values, fibrinogen concentrations in group H decreased at 15 min, 3 h and 6 h after H-LR, but increased to double that of the baseline value at 24 h; platelet counts decreased throughout the study; clot strength was decreased at 15 min, 3 h and 6 h, but returned to baseline value at 24 h after H-LR. Hemorrhage caused decreases in fibrinogen and platelets, and compromised clot strength. The rebound of fibrinogen at 24 h restored clot strength despite platelet deficit. These data suggest the potential compensatory role of fibrinogen in restoring coagulation function in vivo after hemorrhagic shock.     

A new model of severe hemorrhagic shock in rats

We here introduce a fixed-pressure model of hemorrhagic shock in rats that maximizes effects on mean arterial blood pressure (MAP) during shock and yet maintains high reproducibility and controllability. The MAP of rats was adjusted to 25 to 30 mm Hg by blood withdrawals during 30 min. After a shock period of 60 min, rats were resuscitated either with lactated Ringer solution (LR) only or with the collected blood 3-fold diluted with LR (LR + blood) and monitored for further 150 min. Throughout the experiment, vital parameters and plasma marker enzyme activities and creatinine concentration were assessed. Thereafter, liver, kidneys, small intestine, heart, and lung were harvested and evaluated histopathologically. Vital parameters, plasma marker enzyme activities, creatinine concentration, and histopathology indicated pronounced but reliable and reproducible systemic effects and marked organ damage due to hemorrhagic shock and resuscitation. In contrast to rats that received LR + blood, which survived the postresuscitation period, rats receiving LR only invariably died shortly after resuscitation. The hemorrhagic shock model we present here maximally affects MAP and yet is highly reproducible in rats, allowing the study of various aspects of hemorrhagic shock and resuscitation under clinically relevant conditions.     

Hemorrhage induces an increase in serum TNF which is not associated with elevated levels of endotoxin

Although tumor necrosis factor (TNF) and interleukin 6 (IL 6) are purported to be important mediators of inflammatory responses following trauma, it is not known if the serum levels of these cytokines are altered by simple hemorrhage. The objective of this study therefore was to determine whether or not: 1) there is any elevation of TNF or IL 6, and 2) if endotoxin, an important upregulator of these cytokines, is also increased following hemorrhage. To study this, C3H/HeN mice were bled to, and maintained at a mean blood pressure of 35 mmHg for 60 min, and then resuscitated with their own shed blood and adequate fluid. Mice were sacrificed at 30 min into hemorrhage and at 2, 4 or 24 hr post-hemorrhage to obtain serum samples. IL 6 and TNF levels were measured using cytokine dependent cellular assays. Using a quantitative Limulus amebocyte lysate assay, endotoxin levels were determined. TNF levels were significantly elevated at 30 min into hemorrhage, remaining so at 2 hr after resuscitation, but absent by 4 hr. Although there was a trend toward elevated IL 6 levels at 2 hr following hemorrhage, which was sustained up to 24 hr, the values were not significantly different from sham controls. When compared to controls, no marked increase in endotoxin was seen at any time point during or following hemorrhage. These results indicate that hemorrhage, in the absence of significant tissue trauma, causes enhanced TNF release which is not the result of increased endotoxin.     

High-volume ventilation induces pentraxin 3 expression in multiple acute lung injury models in rats

Pentraxin 3 (PTX3) is an acute-phase protein, which can be produced by a variety of tissue cells at the site of infection or inflammation. It plays an important role in innate immunity in the lung and in mediating acute lung injury. The aim of this study was to determine the effect of mechanical ventilation on PTX3 expression in multiple lung injury models. Male Sprague-Dawley rats were challenged with intravenous injection of lipopolysaccharide (LPS) or hemorrhage followed by resuscitation (HS). The animals were then subjected to either relatively higher (12 ml/kg) or lower (6 ml/kg, positive end-expiratory pressure of 5 cmH(2)O) volume ventilation for 4 h. High-volume ventilation significantly enhanced PTX3 expression in the lung, either alone or in combination with LPS or hemorrhage. A significant increase of PTX3 immunohistochemistry staining in the lung was seen in all injury groups. The PTX3 expression was highly correlated with the severity of lung injury determined by blood gas, lung elastance, and wet-to-dry ratio. To determine the effects of HS, LPS, or injurious ventilation (25 ml/kg) alone on PTX3 expression, another group of rats was studied. Injurious ventilation significantly damaged the lung and increased PTX3 expression. A local expression of PTX3 induced by high-volume ventilation, either alone or in combination with other pathological conditions, suggests that it may be an important mediator in ventilator-induced lung injury.     

Molecular basis of protective effect by crocetin on survival and liver tissue damage following hemorrhagic shock

Hemorrhagic shock (HS) causes reduction of cellular energy stores, as measured by levels of ATP and ADP. Furthermore, energy depletion may cause mitochondrial damage, which in turn leads to cell death by apoptosis. The hypothesis of the present study is that by enhancing the recovery of cellular ATP and ADP and mitochondrial damage can be reduced, and the extent of apoptosis minimized. Crocetin, a carotenoid compound, appears to enhance the diffusion of oxygen in aqueous solution, and hence may improve energy stores both to the cell and within it. HS was produced in Sprague–Dawley rats by withdrawing blood from the carotid cannula until a mean arterial pressure of 35–40 mm Hg was reached, and then maintained by further withdrawals of blood for 30 and 60 min. Crocetin was administered 2–4 mg/kg in resuscitation fluid through venus cannula and the animals survived for 24–48 h after HS. Experiments designed to promote tissue reconstitution of ATP using crocetin indicate that these approaches are successful in increasing ATP post-hemorrhage and survival. Crocetin treatment also inhibited cellular damage as indicated by increase of Bcl-2 following decrease in cytosolic cytochrome c and caspase-3 after resuscitation. The prolonged energy deficit seen after hemorrhagic shock can produce late damage and rapid restoration of ATP levels to baseline can reduce apoptosis. In conclusions, crocetin can minimize the cellular damage as evidenced by apoptosis and increased the survival of rats. (Mol Cell Biochem 278: 139–146, 2005)     

Geldanamycin inhibits hemorrhage-induced increases in caspase-3 activity: role of inducible nitric oxide synthase.

Hemorrhage has been shown to increase inducible nitric oxide synthase (iNOS) and deplete ATP levels in tissues and geldanamycin limits both processes. Moreover, it is evident that inhibition of iNOS reduces caspase-3 and increases survival. Thus we sought to identify the molecular events responsible for the beneficial effect of geldanamycin. Hemorrhage in mice significantly increased caspase-3 activity and protein while treatment with geldanamycin significantly limited these increases. Similarly, geldanamycin inhibited increases in proteins forming the apoptosome (a complex of caspase-9, cytochrome c, and Apaf-1). Modulation of the expression of iNOS by iNOS gene transfection or siRNA treatment demonstrated that the level of iNOS correlates with caspase-3 activity. Our data indicate that geldanamycin limits caspase-3 expression and protects from organ injury by suppressing iNOS expression and apoptosome formation. Geldanamycin, therefore, may prove useful as an adjuvant in fluids used to treat patients suffering blood loss.     

Maintenance of lung myeloperoxidase activity in proestrus females after trauma-hemorrhage: upregulation of heme oxygenase-1

Previous studies showed that females in the proestrus stage of the reproductive cycle maintain organ functions after trauma-hemorrhage. However, it remains unknown whether the female reproductive cycle is an important variable in the regulation of lung injury after trauma-hemorrhage and, if so, whether the effect is mediated via upregulation of heme oxygenase (HO)-1. To examine this, female Sprague-Dawley rats during diestrus, proestrus, estrus, and metestrus phases of the reproductive cycle or 14 days after ovariectomy underwent soft tissue trauma and then hemorrhage (mean blood pressure 40 mmHg for 90 min followed by fluid resuscitation). At 2 h after trauma-hemorrhage or sham operation, lung myeloperoxidase (MPO) activity and intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and HO-1 protein levels were measured. Plasma 17beta-estradiol concentration was also determined. The results indicated that trauma-hemorrhage increased lung MPO activity and ICAM-1, CINC-1, and CINC-3 levels in ovariectomized females. These parameters were found to be similar to sham-operated animals in proestrus female rats subjected to trauma-hemorrhage. Lung HO-1 protein level in proestrus females was increased significantly compared with female rats subjected to trauma-hemorrhage during diestrus, estrus, and metestrus phases of the reproductive cycle and ovariectomized rats. Furthermore, plasma 17beta-estradiol level was highest in proestrus females. Administration of the HO inhibitor chromium mesoporphyrin prevented the attenuation of shock-induced lung damage in proestrus females. Thus these findings suggest that the female reproductive cycle is an important variable in the regulation of lung injury following trauma-hemorrhage and that the protective effect in proestrus females is likely mediated via upregulation of HO-1.     

Geldanamycin treatment inhibits hemorrhage-induced increases in KLF6 and iNOS expression in unresuscitated mouse organs: role of inducible HSP70.

The aim of this study was to determine whether hemorrhage affects the levels of a variety of stress-related proteins and whether changes can be inhibited by drugs reported to provide protection from ischemia and reperfusion injury. Male Swiss Webster mice were subjected to a 40% hemorrhage without resuscitation. Western blot analysis indicated that c-Jun (an AP-1 protein), Kruppel-like factor 6 (KFL6), and inducible nitric oxide synthase (iNOS) were upregulated sequentially in that order. Pretreatment of mice with geldanamycin (GA) 16 h before hemorrhage effectively inhibited the expression of the proteins KLF6 and iNOS, whereas caffeic acid phenethyl ester did not. GA pretreatment increased inducible heat shock protein (HSP) 70 but not HSP90 in both sham and hemorrhagic tissues. The overexpressed inducible HSP70 formed complexes with KLF6 and iNOS. These results suggest that GA may be therapeutically useful for reducing hemorrhage-induced injury when used as a presurgical treatment or when added to resuscitation fluids.     

Time course of increased heme oxygenase activity and expression after experimental intracerebral hemorrhage: correlation with oxidative injury

Heme oxygenase (HO) activity in tissue adjacent to an intracerebral hematoma may modulate cellular vulnerability to heme-mediated oxidative injury. Although HO-1 is induced after experimental intracerebral hemorrhage (ICH), the time course of this induction, its effect on tissue HO activity, and its association with oxidative injury markers has not been defined. We therefore quantified HO activity, HO-1 expression, tissue heme content, and protein carbonylation for 8 days after injection of autologous blood into the mouse striatum. Increased striatal HO-1 protein was observed within 24 h, peaked on day 5 at a level that was 10-fold greater than baseline, and returned to baseline by day 8; HO-2 expression was not altered. HO activity increased by only 1.6-fold at its peak on day 5, and had also returned to baseline by day 8. A significant increase in protein carbonylation was observed at 3–5 days, which also was markedly attenuated by 8 days, concomitant with a return of tissue heme to near-normal levels. These results suggest that the increase in HO activity in tissue surrounding an experimental ICH is considerably less than would be predicted based on an analysis of HO-1 expression per se . As HO-1 expression is temporally associated with increased tissue heme and increased protein carbonylation, it may be more useful as a marker of heme-mediated oxidative stress in ICH models, rather than as an index of HO activity.     

Role of PPARγ in the salutary effects of 17β‐estradiol on kupffer cell cytokine production following trauma‐hemorrhage

Studies have shown that administration of 17β‐estradiol prevents trauma‐hemorrhage‐induced increase in proinflammatory cytokine production by Kupffer cells and associated multiple organ injury. Since activation of peroxisome proliferator‐activated receptor γ (PPARγ) following ischemic conditions has been shown to be protective, we examined if PPARγ plays any role in the salutary effects of 17β‐estradiol on Kupffer cell cytokine production following trauma‐hemorrhage. Male mice underwent trauma‐hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). 17β‐estradiol (50 µg/kg) or vehicle with or without PPARγ antagonist GW9662 was injected subcutaneously at the middle of resuscitation. At 2 h after trauma‐hemorrhage, plasma interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α levels, Kupffer cell IL‐6 and TNF‐α production and mRNA expression, and PPARγ, nuclear factor (NF)‐κB and activator protein (AP)‐1 DNA binding activity were determined. Kupffer cell IL‐6 and TNF‐α production, as well as plasma IL‐6 and TNF‐α levels, increased following trauma‐hemorrhage. Moreover, NF‐κB and AP‐1 DNA binding activity and IL‐6 and TNF‐α mRNA expression were also enhanced under such conditions. However, 17β‐estradiol administration normalized all these parameters. Although PPARγ activity decreased after trauma‐hemorrhage, administration of 17β‐estradiol following trauma‐hemorrhage elevated PPARγ activity above the normal level. Inhibition of PPARγ by co‐administration of GW9662, however, abolished the salutary effects of 17β‐estradiol on plasma cytokine and Kupffer cells. Thus, activation of PPARγ appears to play an important role in mediating the salutary effects of 17β‐estradiol on plasma cytokine levels and Kupffer cell cytokine production after trauma‐hemorrhage, which are likely mediated via NF‐κB and AP‐1. J. Cell. Physiol. 226: 205–211, 2010. © 2010 Wiley‐Liss, Inc.     

Testosterone receptor blockade after trauma and hemorrhage attenuates depressed adrenal function

Although the testosterone receptor antagonist flutamide restores the depressed immune function in males after trauma and hemorrhage, it remains unknown whether this agent has any salutary effects on adrenal function. To study this, male rats underwent laparotomy and were bled to and maintained at a blood pressure of 40 mmHg until 40% of the shed blood volume was returned in the form of Ringer lactate. Animals were then resuscitated and flutamide (25 mg/kg body wt) was administered subcutaneously. Plasma adrenocorticotropic hormone (ACTH) and corticosterone, as well as adrenal corticosterone and cAMP were measured 20 h after resuscitation. In additional animals, ACTH was administered and ACTH-induced corticosterone release and adrenal cAMP were determined. The results indicate that adrenal contents of corticosterone and cAMP were significantly decreased and morphology was altered after hemorrhage. Administration of flutamide improved corticosterone content, restored cAMP content, and attenuated adrenal morphological alterations. Flutamide also improved the diminished ACTH-induced corticosterone release and adrenal cAMP response at 20 h after hemorrhage and resuscitation. Furthermore, the diminished corticosterone response to ACTH stimulation in the isolated adrenal preparation was improved with flutamide. These results suggest that flutamide is a useful adjunct for improving adrenal function in males following trauma and hemorrhage.     

Caspase inhibition reduces cardiac myocyte dyshomeostasis and improves cardiac contractile function after major burn injury.

In the heart, thermal injury activates a group of intracellular cysteine proteases known as caspases, which have been suggested to contribute to myocyte inflammation and dyshomeostasis. In this study, Sprague-Dawley rats were given either a third-degree burn over 40% total body surface area plus conventional fluid resuscitation or sham burn injury. Experimental groups included 1) sham burn given vehicle, 400 microl DMSO; 2) sham burn given Q-VD-OPh (6 mg/kg), a highly specific and stable caspase inhibitor, 24 and 1 h prior to sham burn; 3) burn given vehicle, DMSO as above; 4) burn given Q-VD-OPh (6 mg/kg) 24 and 1 h prior to burn. Twenty-four hours postburn, hearts were harvested and studied with regard to myocardial intracellular sodium concentration, intracellular pH, ATP, and phosphocreatine (23Na/31P nuclear magnetic resonance); myocardial caspase-1, -3,and -8 expression; myocyte Na+ (fluorescent indicator, sodium-binding benzofurzan isophthalate); myocyte secretion of TNF-alpha, IL-1beta, IL-6, and IL-10; and myocardial performance (Langendorff). Burn injury treated with vehicle alone produced increased myocardial expression of caspase-1, -3, and -8, myocyte Na+ loading, cytokine secretion, and myocardial contractile depression; cellular pH, ATP, and phosphocreatine were stable. Q-VD-OPh treatment in burned rats attenuated myocardial caspase expression, prevented burn-related myocardial Na+ loading, attenuated myocyte cytokine responses, and improved myocardial contraction and relaxation. The present data suggest that signaling through myocardial caspases plays a pivotal role in burn-related myocyte sodium dyshomeostasis and myocyte inflammation, perhaps contributing to burn-related contractile dysfunction.