Supernatant of traumatized muscle induces inflammation and pain, but not microcirculatory perfusion failure and apoptotic cell death

Soft tissue trauma induces an inflammatory response locally and in remote organs. Although remote organ failure is attributed to the systemic action of locally released mediators, it is so far unclear to what extent a direct cell injury and the consequences of ischemia or a secondary injury due to locally released mediators contribute to the manifestation of tissue damage at the primary site of trauma. Soft tissue trauma was induced by means of a controlled impact injury technique in the hind limb of pentobarbital-anesthetized rats. Additional animals received a femoral arterial infusion of supernatant of traumatized muscle tissue, of nontraumatized muscle, or 0.9% NaCl. Tissue injury was assessed by determining microcirculatory perfusion failure, inflammatory response, apoptotic cell death, and nociceptive pain behavior. Muscle tissue of traumatized animals revealed perfusion failure, tissue hypoxia, and inflammation. Nociceptive testing showed a decrease in mechanical pain thresholds of the affected hind paw. Infusion of supernatant of traumatized tissue induced local inflammation and pain comparable with that of directly traumatized tissue; however, it failed to cause nutritive perfusion failure. Supernatant of nontraumatized muscle did not affect muscle microcirculation and integrity. Only animals that underwent direct trauma presented with apoptotic cell death, as given by in vivo fluorescence microscopy, caspase 3 protein cleavage, and transferase-mediated dUTP nick-end labeling histology. Trauma-associated humoral factors cause post-traumatic hyperalgesia and inflammation, but not microvascular perfusion failure and apoptotic cell death. This finding may prompt future efforts in the therapy of closed soft tissue trauma to focus not only on antimediator strategies, but to add regimens targeting perfusion failure and tissue apoptosis.     

Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia

OBJECTIVE: To characterize microcirculatory actions of activated protein C in an endotoxemia rodent model that allows in vivo studies of microvascular inflammation and perfusion dysfunction. DESIGN: Animal study using intravital microscopy. SETTING: Animal research facility. SUBJECTS: Male Syrian golden hamsters, 6-8 wks old with a body weight of 60-80 g. INTERVENTIONS: In skinfold preparations, endotoxemia was induced by intravenous administration of 2 mg/kg endotoxin (lipopolysaccharide, Escherichia coli). Intravital microscopy allowed quantitative analysis of arteriolar and venular leukocyte adhesion and functional capillary density (cm) that served as a measure of microvascular perfusion failure. Activated protein C (APC group, n = 8, 24 microg/kg intravenously) was substituted continuously during 8 hrs after lipopolysaccharide, whereas endotoxemic buffer-treated animals (control, n = 7) served as controls. MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide increased leukocyte adhesion and decreased functional capillary density to 50% of baseline values (p <.01 vs. baseline). Activated protein C treatment inhibited (p <.05) lipopolysaccharide-mediated leukocytic response and attenuated (p <.05) endotoxic perfusion failure in nutritive capillaries. CONCLUSIONS: Activated protein C-induced protection from lipopolysaccharide-mediated microcirculatory dysfunction was characterized in vivo for the first time. The impressive modification of leukocyte cross-talk indicates systemic anti-inflammatory activated protein C effects on leukocytes and the endothelium, subsequently improving capillary perfusion. These actions could represent the in vivo mechanism of activated protein C interactions observed in patients with severe sepsis.     

Development of an acute burn model in adult mice for studies of cardiac function and cardiomyocyte cellular function

The increasing availability of mice with gene supplementation (transgenic), site-specific inactivation mutations (gene "knock-outs"), or site-specific genetic modification mutations (gene "knock-ins") has spurred interest in the development of murine trauma models. In this study, C57 BL/6 mice (28 g) were given a cutaneous burn over 40% total body surface area by applying brass probes (1 x 2 x 0.003 cm) heated to 100 degrees C in boiling water to the animals side and back for 5 s. Shams received anesthesia alone and not burn. Mice were killed 24 h post-burn to determine presence of partial-thickness or full-thickness burn injury, cardiac contractile function (Langendorff perfusion, n = 7 or 8 mice/group) or to examine cardiac myocyte cytokine secretion in isolated cardiomyocytes (collagenase perfusion, n = 4 or 5 mice/group). All mice were killed 24 h post-burn for subsequent cardiac or cardiomyocyte studies. Our studies confirm that this murine model of burn trauma produced mixed partial- or full-thickness burn injury, whereas there was no necrosis or inflammation in sham burn mice. Baseline hematocrits were similar in all mice (44+/-1) but decreased after burn trauma (37+/-1), likely because of the volume of fluid resuscitation and hemodilution. Burn trauma impaired cardiac contraction and relaxation as indicated by the lower left ventricular pressure (LVP) measured in burn (56+/-4) compared to that measured in shams (84+/-1 mmHg, P < 0.001), a lower rate of LVP rise (+dP/dt max, 1393+/-10 vs. 2000+/-41 mmHg/s, P < 0.002), and reduced LVP fall (-dP/dt max, 1023 - 40 vs. 1550+/-50, P < 0.001). These differences occurred despite similar coronary perfusion pressures and heart rates in both sham and burn mice. Ventricular function curves were shifted downward in the burn mice in the direction of contractile failure; in addition, hearts from burn mice had reduced LVP and +dP/dt responses to increases in coronary flow rate, increases in perfusate Ca2+, and to isoproterenol challenge (P < 0.05). Burn trauma promoted cardiac myocyte secretion of tumor necrosis factor (TNFalpha) (175+/-6 pg/mL) compared to that measured in shams (72+/-9 pg/mL, P < 0.05); burn trauma also increased cardiac myocyte secretion of interleukin 1beta (IL-1beta) (sham: 2+/-0.5; burn: 22+/-1 pg/mL, P < 0.05) and IL-6 (sham: 70+/-6; burn: 148+/-16 pg/mL, P < 0.05). Anti-TNFalpha strategies prevented burn-mediated cardiac contractile deficits. Burn trauma altered Ca2+ homeostasis in murine cardiomyocytes (Fura-2 AM loading). [Ca2+]i in myocytes from burns (185+/-4 nM) was higher than values measured in myocytes from shams (86+/-nM, P < 0.05). These data confirm that the murine burn model provides a reasonable approach to study the molecular and cell biology of inflammation in organ dysfunction after burn trauma.     

The impact of the Micro-Lightguide O2C for the quantification of tissue ischemia in diabetic foot ulcers

OBJECTIVE: Tissue oxygen supply is crucial for wound healing. Especially in diabetic foot lesions, the chances for healing are mainly dependent on the presence or absence of ischemia. This study investigates the impact of the tissue O2 analysis system "O2C" for noninvasive quantification of tissue oxygenation in diabetic foot ulcer patients. RESEARCH DESIGN AND METHODS: O2C assessed relative blood flow (flow), flow velocity (velo), and hemoglobin concentration (rHb) and hemoglobin oxygenation (SO2) at 2 and 6 mm depth (means +/- SE). 1) Measurements were performed on intact skin of the forefoot and forearm of 20 healthy volunteers on 2 consecutive days. 2) Parameters were assessed on intact skin of the forefoot of diabetic foot ulcer patients (n=14). 3) Measurements were performed directly at the wound site in diabetic patients (n = 14). RESULTS: 1) Flow, velo, rHb, and SO2 at 2 and 6 mm depth were not significantly different when measured at 2 consecutive days. 2) There were no significant differences between diabetic subjects and healthy volunteers. Only flow in 6 mm depth was significantly higher in diabetic subjects (75 +/- 13 vs. 51 +/- 0.4 arbitrary units [AU], P <0.05). When diabetic foot ulcer patients were split into healers and nonhealers, initial readings of SO2 at 2 mm (32 +/- 6 vs. 44 +/- 3%, P <0.05) and flow in 6 mm (28 +/- 1 vs. 51 +/- 0.6 AU, P <0.05) were significantly reduced in nonhealers compared with control subjects, whereas in healers flow in 6 mm (70 +/- 0.6 vs. 51 +/- 0.6 AU, P <0.05) was significantly higher than that in control subjects. 3) Initial SO2, rHb, flow, and velo were significantly lower in nonhealing compared with healing wounds. CONCLUSIONS: O2C is a new reliable and valid method for noninvasive measurement of tissue oxygenation and microvascular blood flow in patients with diabetic foot ulcers.     

Effect of systemic hypothermia on local soft tissue trauma-induced microcirculatory and cellular dysfunction in mice

OBJECTIVE: Changes in body temperature occur as a systemic reaction to severe trauma; however, its role in the manifestation of injury remains unclear. Thermoregulatory responses vary considerably from fever to hypothermia. Although hypothermic trauma patients seem to have a worse prognosis, there is the question whether hypothermia per se or the severity of trauma producing the hypothermia is responsible for aggravated injury and increased mortality rate. The present study unravels how moderate to severe systemic hypothermia modulates local microcirculatory dysfunction and cellular injury in local soft tissue trauma. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university. SUBJECTS: C57BL/6J mice. INTERVENTIONS: A model involving standardized drop weight device-induced tissue trauma and high-resolution multifluorescence microscopy in the dorsal skinfold chamber was used to show arteriolar vasoconstriction, reduction of blood flow, nutritive perfusion failure, and apoptotic cell death at 1 hr after trauma. MEASUREMENTS AND MAIN RESULTS: During the 8-hr posttrauma observation period, microcirculation, but not apoptosis, restituted to almost baseline level. Concomitant systemic hypothermia of either 34 degrees C or 30 degrees C did not affect late manifestation of apoptotic cell death but aggravated initial microcirculatory dysfunction and inhibited recovery during the 8-hr follow-up period. CONCLUSIONS: Our study provides evidence that systemic hypothermia may aggravate soft tissue trauma-associated microcirculatory dysfunction. These experimental results clearly support clinical efforts to prevent hypothermia in the acutely traumatized patient.     

Liposome-encapsulated hemoglobin does not exacerbate endotoxin-induced lung injury

OBJECTIVE: To test the hypothesis that liposome encapsulated hemoglobin (LEH), an experimental oxygen-carrying fluid, exacerbates endotoxin-induced lung injury in the rat. DESIGN: Prospective, randomized animal study. SETTING: University animal laboratory. METHODS: Anesthetized Sprague-Dawley rats (n = 8-13) were infused with LEH (10% of estimated total blood volume) or vehicle (0.9% NaCl). Thirty minutes later, Escherichia coli endotoxin (3.6 mg/kg, i.v.) or vehicle (0.9% NaCl) was administered, and skeletal muscle oxygen tension as well as lung injury were assessed at 2, 4, and 8 hrs. Oxygen tension was measured using a miniaturized thin film oxygen sensor placed in the rectus abdominis muscle, and lung injury was evaluated by determining lung weights, lung myeloperoxidase activity, lung tissue tumor necrosis factor-alpha level, and protein concentration in bronchoalveolar lavage fluid. RESULTS: The intravenous bolus injection of E. coli endotoxin elevated lung water content (33% +/- 5%; p < .01 vs. sham controls), myeloperoxidase activity (56% +/- 6%; p < .01), and tumor necrosis factor-alpha production (1320 +/- 154 pg/g lung tissue; p < .05 vs. undetected levels in sham controls), as well as induced protein accumulation in bronchoalveolar lavage fluid (258% +/- 38%; p < .01) and skeletal muscle hypoxia (52 +/- 8 mm Hg; p < .05). Pretreatment with LEH, which when infused alone did not induce lung injury, had no effect on these responses. CONCLUSION: In this specific model of endotoxin-induced lung injury, LEH does not exacerbate microvascular leakage and leukosequestration, the hallmarks of adult respiratory distress syndrome.     

Reduction in intestinal leukocyte adherence in rat experimental endotoxemia by treatment with the 21-aminosteroid U-74389G

OBJECTIVES: To investigate leukocyte adherence in intestinal venules in experimental endotoxemia after treatment with the 21-aminosteroid U-74389G. DESIGN AND SETTING: Prospective, randomized, controlled animal study in an experimental laboratory. SUBJECTS: Twenty-one male Wistar rats weighing 190 +/- 40 g. INTERVENTIONS: The rats were divided equally into three groups: (a) control group, (b) endotoxemia (5 mg/kg lipopolysacharide from Escherichia coli O55:B5), and (c) endotoxemia and U-74389G administration 30 min before (3 mg/kg) and 60 min after endotoxin challenge (1.5 mg/ kg). MEASUREMENTS AND MAIN RESULTS: The distal small intestine of the animals was examined using intravital fluorescence videomicroscopy 2 h after endotoxin challenge. Leukocytes were stained in vivo by means of rhodamine 6G. In the endotoxemic animals we observed a fourfold increase in the count of firmly adherent leukocytes in submucosal post-capillary and collecting venules. Treatment with the 21-aminosteroid U-74389G significantly attenuated the count of sticking leukocytes in the collecting venules (control, 61 +/- 10 cells/mm2; lipopolysaccharide, 237 +/- 42 cells/mm2; U-74389G 125 +/- 9 cells/mm2; p < 0.05). In these venules leukocyte rolling behavior was comparable to that in the control group without endotoxin challenge. CONCLUSIONS: Administration of U-74389G, which has radical scavenging properties, attenuates leukocyte adherence in selected populations of intestinal venules which is found increased during endotoxemia. Thus, 21-aminosteroids may have an impact in the treatment of endotoxin-induced intestinal injury.     

Endotoxemia inhibits intestinal adaptation in a rat model of short bowel syndrome

Sepsis is commonly associated with or complicates short bowel syndrome (SBS). The purpose of the present study was to investigate the effects of endotoxemia on intestinal adaptation in a rat model of SBS. Male Sprague-Dawley rats were divided into three experimental groups: Sham rats underwent bowel transection and re-anastomosis, SBS rats underwent 75% small bowel resection, and SBS-LPS rats underwent bowel resection and were given lipopolysaccharide. Bowel weight, organ weights, and parameters of intestinal adaptation (bowel and mucosal weights, mucosal DNA and protein, villus height, and crypt depth) were determined on day 15 following operation. The results of this study demonstrate that SBS rats showed a significant increase (vs. Sham) in jejunal and ileal bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth. SBS-LPS animals demonstrated lower (vs. SBS rats) final body weight (215 +/- 7 vs. 287 +/- 10 g, P < 0.05), overall weight in duodenum (98+/- 2 vs. 119 +/-5 mg/cm, P < 0.05) and jejunum (144 +/- 9 vs. 189 +/- 16 mg/cm, P < 0.05), mucosal weight in jejunum (54 +/- 5 vs. 69 +/- 5 mg/cm, P < 0.05) and ileum (31 +/- 2 vs. 37 +/- 3 mg/cm, P < 0.05), mucosal DNA in jejunum (89 +/- 11 vs. 120 +/- 11 microg/cm, P < 0.05) and ileum (46 +/- 6 vs. 61 +/- 4 microg/cm, P < 0.05), jejunal crypt depth (152 +/- 19 vs. 189 +/- 12 microm, P < 0.05), and ileal villus height (405 +/- 63 vs. 515 +/- 30 pm, P < 0.05). In addition, the SBS group had no late (second week) mortality, whereas the SBS-LPS group had an 17% late mortality rate. In conclusion, in a rat model of SBS-LPS, endotoxemia appears to inhibit structural intestinal adaptation and increase mortality.     

Randomized, open label, controlled clinical trial of oral administration of an egg albumin-based protein supplement to patients on continuous ambulatory peritoneal dialysis.

BACKGROUND/AIM: Malnutrition is highly prevalent in patients on continuous ambulatory peritoneal dialysis (CAPD) and is a strong predictor of increased morbidity and mortality. Therefore, the aim of this study was to evaluate the effect of oral administration of an egg albumin-based protein supplement on the nutritional status of CAPD patients. METHODS: In this randomized, open label, controlled clinical trial, 28 CAPD patients were allocated to a study (n = 13) or a control (n = 15) group. Both groups received conventional nutritional counseling; the study group received, additionally, an oral egg albumin-based supplement. During a 6-month follow-up, all patients had monthly clinical and biochemical evaluations and quarterly assessments of adequacy of dialysis and nutrition. RESULTS: Serum albumin Levels were not different between groups; however, a significant increase (baseline vs final) was observed in the study group (2.64+/-0.35 vs 3.05+/-0.72 g/dL) but not in the control group (2.66+/-0.56 vs 2.80+/-0.54 mg/dL). Calorie and protein intake increased more in the study group (calories 1331+/-432 vs 1872+/-698 kcal; proteins 1.0+/-0.3 vs 1.7+/-0.7 g/kg) than in the control group (calories 1423+/-410 vs 1567+/-381 kcal; proteins 1.0+/-0.4 vs 1.0+/-0.3 g/kg). Similarly, non-protein nitrogen appearance rate (nPNA) increased significantly more in the study (1.00+/-0.23 vs 1.18+/-0.35 g/kg/day) than in the control group (0.91+/-0.11 vs 0.97+/-0.14 g/kg/ day). Triceps skinfold thickness (TSF) and midarm muscle area (MAMA) displayed a nonsignificant trend to a greater increase in the study group (TSF 16.7+/-8.7 vs 18.3+/-10.7 mm; MAMA 23.8+/-6.2 vs 25.8+/-5.9 cm2) than in controls (TSF 16.4+/-5.7 vs 16.9+/-7.0 mm; MAMA 28.7+/-7.8 vs 30.0+/-7.9 cm2). At the end of follow-up, the frequency of patients with moderate or severe malnutrition decreased 6% in the control group and decreased 28% in the study group. At the final evaluation, the most important predictors of serum albumin were the oral egg albumin-based supplement administration and protein intake (p < 0.05); secondary predictors (p = 0.06) were peritoneal transport rate and MAMA. CONCLUSIONS: In the study group, oral administration of the egg albumin-based supplement significantly improved serum albumin, calorie and protein intake, and nPNA, and, compared to controls, this maneuver was associated with a trend to increased anthropometric parameters and improved Subjective Global Assessment evaluation. Oral administration of the albumin supplement and protein intake were the most significant predictors of serum albumin at the end of follow-up. This oral supplement may be a safe, effective, and cheap method to improve nutritional status in peritoneal dialysis patients.     

Leukocyte-independent plasma extravasation during endotoxemia

OBJECTIVES: To determine the meaning of leukocyte-endothelial interactions for the development of endotoxin-induced vascular leakage. DESIGN: Randomized, blinded, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Twenty-four male Wistar rats. INTERVENTIONS: After application of fucoidin to prevent leukocyte rolling and adherence (25 mg/kg; n = 8; fucoidin/LPS group) or saline 0.9% (n = 8; LPS group), animals were given an intravenous infusion of endotoxin (Escherichia coli lipopolysaccharide 026:B6; 2 mg/kg/hr) over 120 mins. Animals in the control group (n = 8) received an equivalent volume of saline 0.9%. MEASUREMENTS AND MAIN RESULTS: Leukocyte rolling and leukocyte adherence, red cell velocity, vessel diameters, venular wall shear rate, volumetric blood flow, and macromolecular leakage were determined in mesenteric postcapillary venules using in vivo videomicroscopy at baseline, 60 mins, and 120 mins after start of a continuous endotoxin infusion. Fucoidin prevented leukocyte rolling (baseline, 3+/-2 rollers; 120 mins, 3+/-1 rollers; not significant vs. baseline; p < .01 vs. LPS group) and reduced the adherence of leukocytes at baseline and during endotoxemia and showed only a slight increase in adherent leukocytes (baseline, 100+/-38 cells/mm2; 120 mins, 244+/-68 cells/mm2; p < .05 vs. baseline; p < .01 vs. LPS group). In the LPS group, endotoxin exposure induced a marked increase in adherent leukocytes (baseline, 248+/-24 cells/mm2; 120 mins, 560+/-57 cells/mm2; p < .01). Leukocyte adherence in control animals (control group) did not increase significantly. Macromolecular leakage, expressed as the ratio of perivenular to intravenular fluorescence intensity after injection of fluorescence-labeled albumin, increased from 0.16+/-0.03 to 0.49+/-0.04 (p < .01 vs. baseline; p < .05 vs. control) during the infusion of endotoxin in the LPS group. Fucoidin application did not diminish the extravasation of albumin (baseline, 0.09+/-0.03; 120 mins, 0.61+/-0.10; p < .01 vs. baseline; p < .01 vs. control). CONCLUSIONS: These results demonstrate that despite a significant reduction of adherent leukocytes to the endothelium by fucoidin, there is no reduction in macromolecular leakage, indicating that leukocyte-endothelial interactions only play a minor role for the development of macromolecular leakage and microvascular damage in the early phase of endotoxemia.     

Biologically variable ventilation increases arterial oxygenation over that seen with positive end-expiratory pressure alone in a porcine model of acute respiratory distress syndrome

OBJECTIVES: We compared biologically variable ventilation (BVV) (as previously described) (1) with conventional control mode ventilation (CV) in a model of acute respiratory distress syndrome (ARDS) both at 10 cm H2O positive end-expiratory pressure. DESIGN: Randomized, controlled, prospective study. SETTING: University research laboratory. SUBJECTS: Farm-raised 3- to 4-month-old swine. INTERVENTIONS: Oleic acid (OA) was infused at 0.2 mL/kg/hr with FIO2 = 0.5 and 5 cm H2O positive end-expiratory pressure until PaO2 was < or =60 mm Hg; then all animals were placed on an additional 5 cm H2O positive end-expiratory pressure for the next 4 hrs. Animals were assigned randomly to continue CV (n = 9) or to have CV computer controlled to deliver BVV (variable respiratory rate and tidal volume; n = 8). Hemodynamic, expired gas, airway pressure, and volume data were obtained at baseline (before OA), immediately after OA, and then at 60-min intervals for 4 hrs. MEASUREMENTS AND MAIN RESULTS: At 4 hrs after OA injury, significantly higher PaO2 (213+/-17 vs. 123+/-47 mm Hg; mean+/-SD), lower shunt fraction (6%+/-1% vs. 18%+/-14%), and lower PaCO2 (50+/-8 vs. 65+/-11 mm Hg) were seen with BVV than with CV. Respiratory system compliance was greater by experiment completion with BVV (0.37+/-0.05 vs. 0.31+/-0.08 mL/cm H2O/kg). The improvements in oxygenation, CO2 elimination, and respiratory mechanics occurred without a significant increase in either mean airway pressure (14.3+/-0.9 vs. 14.9+/-1.1 cm H2O) or mean peak airway pressure (39.3+/-3.5 vs. 44.5+/-7.2 cm H2O) with BVV. The oxygen index increased five-fold with OA injury and decreased to significantly lower levels over time with BVV. CONCLUSIONS: In this model of ARDS, BVV with 10 cm H2O positive end-expiratory pressure improved arterial oxygenation over and above that seen with CV with positive end-expiratory pressure alone. Proposed mechanisms for BVV efficacy are discussed.     

Effects of fascia lata on HIFU lesioning in vitro

The effects of fascia lata on high intensity focused ultrasound (US), or HIFU,-induced lesions were demonstrated through comparison with and without fascia lata in bovine thigh muscle tissue. Experiments were conducted in an arrangement with a three-way multiscan ultrasonic inspection system and imaging done by B-mode US. Bovine thigh muscle (8-cm thick) was treated with 1.5 MHz for 8 s. Spatial peak intensity (ISP) was 3000 W/cm2. B-mode US imaging detected appearance at the HIFU treatment site. At a free-field intensity of 4000 W/cm2, the observed lesion length (along the axis) with fascia lata was 12 +/- 1.82 mm, compared with 4 +/- 1.54 mm for samples without fascia lata. At 3000 W/cm2, the values for samples with fascia lata and samples without fascia lata, respectively, were 13 +/- 1.50 mm and 2 +/- 1.42 mm. During a 30-s exposure, at ISP of 2000 W/cm2, the peak temperature reached 41 degrees C in samples without fascia lata and 70 degrees C in samples with fascia lata. At ISP of 3000 and 4000 W/cm2, the peak temperature reached, respectively, 73 degrees C and 84 degrees C in samples without fascia lata, compared with 102 degrees C and 104 degrees C, respectively, for samples with fascia lata. The results confirm that fascia lata contributes to increasing tissue necrosis, temperature elevation and echogenicity in US images.     

Albumin resuscitation improves ventricular contractility and myocardial tissue oxygenation in rat endotoxemia

OBJECTIVE: Fluid resuscitation to improve delivery of oxygen to vital organs is a principal clinical intervention for septic patients. We previously reported that albumin resuscitation in rat endotoxemia improved contractility in isolated cardiomyocytes, but whether this effect occurs in vivo is unknown. We hypothesized that albumin resuscitation would improve decreased ventricular contractility and myocardial tissue oxygenation in vivo. DESIGN: Randomized, controlled, prospective animal study. SETTING: University animal laboratory. SUBJECTS: Male Sprague-Dawley rats (250-350 g). INTERVENTIONS: Rats were randomized into three groups: control with no lipopolysaccharide (n = 8), lipopolysaccharide (10 mg/kg) without albumin resuscitation (n = 8), and lipopolysaccharide with albumin resuscitation (n = 6). Five hours after lipopolysaccharide injection, animals were resuscitated with 10 mL/kg 5% rat albumin in 0.9% saline. Six hours after 10 mL/kg lipopolysaccharide, a pressure-volume conductance catheter (MIKRO-Tip 2.0-Fr, Millar Instruments, Houston, TX) was inserted into the left ventricle to quantify maximum elastance as an index of contractility. Myocardial tissue Po2 was measured using a fiberoptic oxygen probe. MEASUREMENTS AND MAIN RESULTS: Maximum elastance decreased after lipopolysaccharide relative to control (47%, from 5.9 +/- 0.8 to 3.1 +/- 0.4 mm Hg/microL, p < .05). Albumin resuscitation prevented the lipopolysaccharide-induced decrease in maximum elastance (7.0 +/- 1.2 mm Hg/microL, p < .05 vs. lipopolysaccharide). Myocardial tissue Po2 was reduced in endotoxemia compared with control (53%, from 10.1 +/- 0.9 to 4.7 +/- 0.6 mm Hg, p < .05), and albumin resuscitation improved the lipopolysaccharide-induced tissue hypoxia toward the control value (9.0 +/- 1.4 mm Hg, p < .05). CONCLUSIONS: Albumin resuscitation improved decreased ventricular contractility and myocardial oxygenation in endotoxemic rats. This result suggests that albumin resuscitation may improve ventricular dysfunction by improving myocardial hypoxia.     

ATP-induced calcium increase as a potential first signal in mechanical tissue trauma. A laser scanning microscopic study on cultured mouse skeletal myocytes

Although it is known that after major tissue trauma, local incidents in the mechanically destroyed muscle tissue form the basis of subsequently occurring severe inflammatory reactions, the very first events taking place immediately after myocyte destruction have not been studied on the single cell level thus far. Therefore, in this study, the reaction of cultured C2C12 mouse skeletal myocytes to lethal injury was examined using laser scanning microscopy. Mechanical rupture of one single myocyte induced an immediate accumulation of calcium in its cytosol and nuclei, as detected by an increase in the fluorescence intensity of the intracellular calcium-sensitive dye Fluo-3. The intracellular calcium elevation propagated further to the adjacent, noninjured myocytes in a wave-like fashion within seconds. The calcium increase detected in these neighboring cells was higher and up to 1000 times more extended than the physiological calcium spike that induces C2C12 myocyte contraction. Wave propagation did not depend on gap junctional communication but occurred via liberation of nucleotides, mainly ATP, but presumably also UTP and others, from the destroyed cell and subsequent calcium release from the sarcoplasmic reticulum via a purinoceptor-mediated mechanism in the adjacent cells. These findings suggest a decisive role of ATP and related nucleotides in the pathogenesis of tissue trauma because they appear to initiate the signaling mechanism from injured myocytes to the surrounding tissue and potentially to the whole body.     

Involvement of Kupffer cells in the interaction between neutrophils and sinusoidal endothelial cells in rats

During endotoxic liver injury, large numbers of neutrophils infiltrate the liver, and serum levels of tumor necrosis factor-alpha (TNF-alpha) become elevated. The object of this study was to assess the roles of TNF-alpha secreted by Kupffer cells in the interaction between neutrophils and sinusoidal endothelial cells (SECs). Rat neutrophils were perfused onto SECs that were stimulated with either TNF-alpha or supernatant from lipopolysaccharide (LPS)-stimulated Kupffer cells using an in vitro flow system. Numbers of adhered or migrated neutrophils were counted, and the effect of an antibody against intercellular adhesion molecule-1 (ICAM-1) was studied. Compared with controls (200 +/- 21 cells/mm2), neutrophil adhesion to SECs was significantly increased by both TNF-alpha (342 +/- 26 cells/mm2; P < 0.05) and LPS-stimulated Kupffer cell supernatant (331 +/- 29 cells/mm2; P < 0.05). Anti-ICAM-1 significantly inhibited neutrophil adhesion (139 +/- 10 cells/mm2; P < 0.05) and decreased the migration rate of neutrophils on SECs treated with LPS-stimulated Kupffer cell supernatant (P < 0.05). LPS-stimulated Kupffer cells secreted TNF-alpha in an LPS dose-dependent manner, and they significantly enhanced ICAM-1 expression on SECs (P < 0.05 vs. control). In addition, dexamethasone suppressed TNF-alpha production by LPS-stimulated Kupffer cells and decreased ICAM-1 expression and neutrophil adhesion on SECs. These findings suggest that Kupffer cells are involved in neutrophil adhesion and migration in hepatic sinusoids via TNF-alpha production and induction of ICAM-1 expression on SECs during liver injury associated with endotoxemia.     

Tissue oxygenation and perfusion in patients with systemic sepsis.

OBJECTIVE: Multiple organ dysfunction is associated with systemic sepsis. To investigate whether this is attributable to peripheral tissue hypoperfusion and/or cellular hypoxia, simultaneous measurements of tissue perfusion and oxygenation were made in patients with severe sepsis and in controls. DESIGN: Prospective, observational study. SETTING: Adult intensive care unit, tertiary referral center. PATIENTS: Volunteers (group C, n = 7), patients undergoing cardiopulmonary bypass (group B, n = 6), and patients with severe sepsis (group S, n = 6). INTERVENTIONS: Limb ischemia and reperfusion. MEASUREMENTS AND MAIN RESULTS: Tissue oxygenation and microvascular flow were measured by using microelectrodes inserted into brachoradialis muscle and overlying subcutaneous tissue. Forearm cutaneous red cell flux and regional blood flow were measured simultaneously. Responses to 20 mins of limb ischemia and subsequent reperfusion were observed. Baseline muscle tissue oxygenation was greater in sepsis (1.7 +/- 0.2, 1.5 +/- 0.7, and 4.4 +/- 0.6 kPa for groups C, B, and S, respectively, mean +/- sem, p <.05), although baseline subcutaneous tissue oxygenation did not vary between groups. During ischemia tissue oxygenation, values decreased in muscle (to 1.3 +/- 0.2, 1.0 +/- 0.4, and 1.5 +/- 0.4 kPa for groups C, B, and S, respectively) and subcutaneous tissue (to 2.0 +/- 0.3, 1.7 +/- 0.5, and 2.3 +/- 0.2 kPa for groups C, B, and S, respectively). Decline in tissue oxygen tension was initially more rapid in septic muscle compared with controls (25% decrease, 68 +/- 23 vs. 176 +/- 38 for group S vs. group C, p <.05, and 50% decrease, 126 +/- 34 vs. 398 +/- 72 secs for group S vs. group C, p <.01). However, overall rate of tissue decline was similar (95% decrease, 444 +/- 122 vs. 614 +/- 96 for group S vs. group C, p >.05). After reperfusion, significant differences in muscle tissue oxygenation reappeared between groups (2.0 +/- 0.3, 1.5 +/- 0.7, and 4.0 +/- 0.4 kPa for groups C, B, and S, respectively, p <.05). There were no differences in time to 25%, 50%, or 95% tissue oxygen recovery. Whole limb reperfusion was significantly less in patient groups compared with controls (10.6 +/- 0.9, 4.5 +/- 1.2, and 4.3 +/- 1.6 mL x 100 mL(-1) x min(-1) for groups C, B, and S, respectively, p <.05). CONCLUSIONS: Significant differences in tissue oxygenation distribution between muscle and subcutaneous tissues occur in patients with severe sepsis. High baseline muscle tissue oxygen levels are accompanied by rapid extraction of oxygen during stagnant ischemia.     

Cerebral response to norepinephrine compared with fluid resuscitation in ovine traumatic brain injury and systemic inflammation

OBJECTIVE: Traumatic brain injury is frequently accompanied by a systemic inflammatory response. Systemic inflammation was associated with cerebral hyperperfusion uncoupled to global oxygen metabolism in ovine head trauma. The present study investigated the cerebral effects of cerebral perfusion pressure (CPP) management performed by either fluid resuscitation or vasopressor treatment of low CPP induced by systemic inflammation. DESIGN: Nonrandomized experimental study. SETTING: University hospital laboratory. SUBJECTS: A total of 12 adult sheep. INTERVENTIONS, MEASUREMENTS, AND MAIN RESULTS: Sheep were anesthetized and ventilated throughout the experimental period (13 hrs). After baseline measurements (hour 0), blunt head trauma was induced by a nonpenetrating stunner. After postinjury measurements (hour 2), all animals received continuous endotoxin infusion. At hour 10, one group (n = 6) was infused with hydroxyethyl starch until CPP reached 60-70 mm Hg. A second group (n = 6) received norepinephrine for CPP elevation. In the norepinephrine group, blood was isovolemically exchanged by hydroxyethyl starch to achieve comparable hematocrit levels. Head trauma increased intracranial pressure and decreased brain tissue oxygen tension. Endotoxemia induced a hyperdynamic cardiovascular response with increased internal carotid blood flow in the presence of systemic hypotension and decreased CPP. Hydroxyethyl starch infusion further increased internal carotid blood flow from (mean +/- sd) 247 +/- 26 (hour 10) to 342 +/- 42 mL/min (hour 13) and intracranial pressure from 20 +/- 4 (hour 10) to a maximum of 25 +/- 3 mm Hg (hour 12) but did not significantly affect brain tissue oxygen tension, sinus venous oxygen saturation and oxygen extraction fraction. Norepinephrine increased internal carotid blood flow from 268 +/- 19 to 342 +/- 58 mL/min and intracranial pressure from 22 +/- 11 to 24 +/- 11 mm Hg (hour 10 vs. hour 13) but significantly increased sinus venous oxygen saturation from 49 +/- 4 (hour 10) to a maximum of 59 +/- 6 mm Hg (hour 12) and decreased oxygen extraction fraction. The increase in brain tissue oxygen tension during norepinephrine treatment was not significant. CONCLUSION: We conclude that despite identical carotid blood flows, only CPP management with norepinephrine reduced the cerebral oxygen deficit in this model.     

Disruption of skeletal myocytes initiates superoxide release: contribution of NADPH oxidase

Generation of reactive oxygen species (ROS) as an early local reaction to muscle crush injury has frequently been predicted. However, although it is known that severe inflammatory reactions occurring after major muscle trauma originate mainly from early local incidents within the injured tissue, no detailed studies exist on the local generation of ROS in response to myocyte destruction thus far. Therefore, in this study, ROS formation after lethal mechanical damage was examined using a model of scraping injury to cultured C2C12 skeletal myocytes and superoxide detection by lucigenin chemiluminescence, nitrotetrazolium blue chloride reduction, or electron spin resonance spectroscopy. Mechanical rupture of myocytes resulted in an immediate release of superoxide from the damaged cells that could be substantially blocked by the superoxide scavengers superoxide dismutase (51%), tiron (95%), and MAMA/NO (93%) and by hypoxia (83% inhibition). Superoxide generation was primarily confined to the myocytes' membrane fraction and 7- to 8-fold enhanced by the addition of NADH or NADPH. The NADPH-enhanced superoxide generation could largely be diminished by the NAD(P)H oxidase inhibitors diphenyleneiodonium and apocynin in cell lysates (97% and 35% inhibition, respectively) and in isolated membrane fractions (61% and 63% inhibition). We thus conclude that immediately after myocyte damage, large amounts of superoxide are formed that predominantly originate from membrane-bound electron-transferring enzymes, especially NAD(P)H oxidase. This suggests a decisive role of ROS in the pathogenesis of tissue trauma, with superoxide being an initiator of the signaling mechanism from injured myocytes to the surrounding tissue and, potentially, to the whole body.     

Calpain inhibition decreases endothelin-1 levels and pulmonary hypertension after cardiopulmonary bypass with deep hypothermic circulatory arrest

OBJECTIVE: Cardiopulmonary bypass in infants and children can result in cardiopulmonary dysfunction through ischemia and reperfusion injury. Pulmonary hypertension and injury are particularly common and morbid complications of neonatal cardiac surgery. Inhibition of calpain, a cysteine protease, has been shown to inhibit reperfusion injury in adult organ systems. The hypothesis is that calpain inhibition can alleviate the cardiopulmonary dysfunction seen in immature animals following ischemia and reperfusion with cardiopulmonary bypass. DESIGN: Animal case study. SETTING: Medical laboratory. SUBJECTS: Crossbred piglets (5-7 kg). INTERVENTIONS: Piglets were cooled with cardiopulmonary bypass to 18 degrees C followed by deep hypothermic circulatory arrest for 120 mins. Animals were rewarmed to 38 degrees C on cardiopulmonary bypass and maintained for 120 mins. Six animals were administered calpain inhibitor (Z-Leu-Leu-Tyr-fluoromethyl ketone; 1 mg/kg, intravenously) 60 mins before cardiopulmonary bypass. Nine animals were administered saline as a control. Plasma endothelin-1, pulmonary and hemodynamic function, and markers of leukocyte activity and injury were measured. MEASUREMENTS AND MAIN RESULTS: Calpain inhibition prevented the increased pulmonary vascular resistance seen in control animals (95.7 +/- 39.4 vs. 325.3 +/- 83.6 dyne.sec/cm, respectively, 120 mins after cardiopulmonary bypass and deep hypothermic circulatory arrest, p = .05). The attenuation in pulmonary vascular resistance was associated with a blunted plasma endothelin-1 response (4.91 +/- 1.72 pg/mL with calpain inhibition vs. 10.66 +/- 6.21 pg/mL in controls, p < .05). Pulmonary function after cardiopulmonary bypass was better maintained after calpain inhibition compared with controls: Po2/Fio2 ratio (507.2 +/- 46.5 vs. 344.7 +/- 140.5, respectively, p < .05) and alveolar-arterial gradient (40.0 +/- 17.2 vs. 128.1 +/- 85.2 mm Hg, respectively, p < .05). Systemic oxygen delivery was higher after calpain inhibition compared with controls (759 +/- 171 vs. 277 +/- 46 mL/min, respectively, p < .001). In addition, endothelial nitric oxide synthase activity in lung tissue was maintained with calpain inhibition. CONCLUSIONS: The reduction in plasma endothelin-1 and maintenance of lung endothelial nitric oxide levels after cardiopulmonary bypass and deep hypothermic circulatory arrest with calpain inhibition were associated with reduced pulmonary vascular resistance. Improved gas exchange and higher systemic oxygen delivery suggest that calpain inhibition may be advantageous for reducing postoperative cardiopulmonary dysfunction commonly associated with pediatric heart surgery and cardiopulmonary bypass.