Mechanical stretch induces lung α-epithelial Na(+) channel expression.

ABSTRACT During fetal development physiological stretching helps drive lung growth and maturation. At birth, the α-subunit of the alveolar epithelial sodium channel (α-ENaC) is a critical factor in helping to facilitate clearance of lung fluid during the perinatal period. The effects of stretch, however, on α-ENaC expression in the fetal lung have yet to be elucidated. In an effort to explore this question, we used both an in vitro cell culture model that exposes cells to repetitive cyclic stretch (CS) as well as an in vivo preterm animal model of mechanical ventilation (MV). We found that murine lung epithelial (MLE-12) cells exposed to repetitive CS showed a significant rise in α-ENaC mRNA expression. Total and cell-surface protein abundance of α-ENaC were also elevated after 24 h of CS. Stretch-induced increases in α-ENaC expression were suppressed in the presence of either actinomycin D or cycloheximide. Pharmacological inhibition of the extracellular signal-regulated protein kinase (ERK1/2) did not attenuate stretch-induced increases in α-ENaC protein, whereas inhibition of p38 MAPK or c-Jun NH2-terminal kinase (JNK) did. In 29-day preterm rabbits, alveolar stretching secondary to postnatal MV markedly elevated fetal lung α-ENaC expression compared to spontaneously breathing counterparts. In summary, our findings indicate that mechanical stretch promotes α-ENaC expression.

Hydroxocobalamin and epinephrine both improve survival in a swine model of cyanide-induced cardiac arrest.

STUDY OBJECTIVE: To determine whether hydroxocobalamin will improve survival compared with epinephrine and saline solution controls in a model of cyanide-induced cardiac arrest. METHODS: Forty-five swine (38 to 42 kg) were tracheally intubated, anesthetized, and central venous and arterial continuous cardiovascular monitoring catheters were inserted. Potassium cyanide was infused until cardiac arrest developed, defined as mean arterial pressure less than 30 mm Hg. Animals were treated with standardized mechanical chest compressions and were randomly assigned to receive one of 3 intravenous bolus therapies: hydroxocobalamin, epinephrine, or saline solution (control). All animals were monitored for 60 minutes after cardiac arrest. Additional epinephrine infusions were used in all arms of the study after return of spontaneous circulation for systolic blood pressure less than 90 mm Hg. A sample size of 15 animals per group was determined according to a power of 80%, a survival difference of 0.5, and an α of 0.05. Repeated-measure ANOVA was used to determine statistically significant changes between groups over time. RESULTS: Baseline weight, time to arrest, and cyanide dose at cardiac arrest were similar in the 3 groups. Coronary perfusion pressures with chest compressions were greater than 15 mm Hg in both treatment groups indicating sufficient compression depth. Zero of 15 (95% confidence interval [CI] 0% to 25%) animals in the control group, 11 of 15 (73%; 95% CI 48% to 90%) in the hydroxocobalamin group, and 11 of 15 (73%; 95% CI 48% to 90%) in the epinephrine group survived to the conclusion of the study (P<.001). The proportion of animals with return of spontaneous circulation at 5 minutes was 4 of 15 (27%; 95% CI 10% to 52%), and that of return of spontaneous circulation at 10 minutes was 11 of 15 (73%; 95% CI 48% to 90%) in the 2 treatment groups. Additional epinephrine infusion after return of spontaneous circulation was administered for hypotension in 2 of 11 (18%; 95% CI 4% to 48%) hydroxocobalamin animals and in 11 of 11 (100%; 95% CI 70% to 100%) of the epinephrine animals (P<.001). At 60 minutes, serum lactate was significantly lower in the hydroxocobalamin group compared with the epinephrine group (4.9 [SD 2.2] versus 12.3 [SD 2.2] mmol/L), and the pH was significantly higher (7.34 [SD 0.03] versus 7.15 [SD 0.07]). Serial blood cyanide levels in the hydroxocobalamin group were also lower than that of the epinephrine group from cardiac arrest through the conclusion of the study. CONCLUSION: Intravenous hydroxocobalamin and epinephrine both independently improved survival compared with saline solution control in our swine model of cyanide-induced cardiac arrest. Hydroxocobalamin improved mean arterial pressure and pH, decreased blood lactate and cyanide levels, and decreased the use of rescue epinephrine therapy compared with that in the epinephrine group.

Hydroxocobalamin and sodium thiosulfate versus sodium nitrite and sodium thiosulfate in the treatment of acute cyanide toxicity in a swine (Sus scrofa) model.

STUDY OBJECTIVE: Cyanide can cause severe hypotension with acute toxicity. To our knowledge, no study has directly compared hydroxocobalamin and sodium nitrite with sodium thiosulfate in an acute cyanide toxicity model. Our objective is to compare the return to baseline of mean arterial blood pressure between 2 groups of swine with acute cyanide toxicity and treated with hydroxocobalamin with sodium thiosulfate or sodium nitrite with sodium thiosulfate. METHODS: Twenty-four swine were intubated, anesthetized, and instrumented (continuous arterial and cardiac output monitoring) and then intoxicated with a continuous cyanide infusion until severe hypotension. The animals were divided into 2 arms of 12 each and then randomly assigned to intravenous hydroxocobalamin (150 mg/kg)+sodium thiosulfate (413 mg/kg) or sodium nitrite (10 mg/kg)+sodium thiosulfate (413 mg/kg) and monitored for 40 minutes after start of antidotal infusion. Twenty animals were needed for 80% power to detect a significant difference in outcomes (alpha 0.05). Repeated measures of analysis of covariance and post hoc t test were used for determining significance. RESULTS: Baseline mean weights, time to hypotension (31 minutes 3 seconds versus 28 minutes 6 seconds), and cyanide dose at hypotension (5.6 versus 5.9 mg/kg) were similar. One animal in the hydroxocobalamin group and 2 animals in the sodium nitrite group died during antidote infusion and were excluded from analysis. Hydroxocobalamin resulted in a faster return to baseline mean arterial pressure, with improvement beginning at 5 minutes and lasting through the conclusion of the study (P<.05). No statistically significant difference was detected between groups for cardiac output, pulse rate, systemic vascular resistance, or mortality at 40 minutes post intoxication. Mean cyanide blood levels (4.03 versus 4.05 microg/mL) and lactate levels (peak 7.9 versus 8.1 mmol/L) at hypotension were similar. Lactate levels (5.1 versus 4.48 mmol/L), pH (7.40 versus 7.37), and base excess (-0.75 versus 1.27) at 40 minutes were also similar. CONCLUSION: Hydroxocobalamin with sodium thiosulfate led to a faster return to baseline mean arterial pressure compared with sodium nitrite with sodium thiosulfate; however, there was no difference between the antidote combinations in mortality, serum acidosis, or serum lactate.

Early versus delayed restoration of flow with temporary vascular shunt reduces circulating markers of injury in a porcine model.

BACKGROUND: Temporary vascular shunting to restore flow after vascular injury has been advocated. The effectiveness of this adjunct in protecting against ischemic injury has not been established. This study will assess the temporal impact of shunts on ischemic injury and arterial flow. METHODS: A porcine model of hind-limb ischemia via iliac artery occlusion was used (N = 36; weight [kg] +/- SD: 89 +/- 4.4). Animals were randomized into one control (Iscctrl) and four study groups (Isc0, Isc1, Isc3, and Isc6) according to ischemic time. Shunt placement followed ischemia, and flow and circulating injury markers were collected incrementally during 18 hours of reperfusion. Flow proportions and a calculated Ischemia Injury Index were used to characterize group differences. RESULTS: There were no intergroup differences concerning initial weight, hemodynamic, or laboratory values. Shunt patency was 92% in the absence of anticoagulation. The proportion of common femoral arterial flow to baseline flow in the Isc6 group was lower than the Iscctrl group (p = 0.02). There was a similar trend with the Isc1 and Isc3 groups. The Ischemia Injury Index demonstrated that there was a difference in the Isc3 and Isc6 groups (late shunt placement) compared with the Iscctrl, Isc0, and Isc1 groups (early shunt placement) (p < 0.001). CONCLUSION: This study provides physiologic insight into the benefit of shunts in a model of extremity ischemia. Early shunting protects the extremity from further ischemic insult and reduces circulating markers of tissue injury. Additionally, the presence of a shunt does not increase the Ischemic Injury Index and patency is maintained in the absence of heparinization.

Predicting irreparable renal ischemic injury using a real-time marker in the porcine model.

PURPOSE: We determined the maximal renal tolerance of warm ischemia using renal cortical interstitial metabolic changes to identify a potential real-time marker of irreparable renal function. MATERIALS AND METHODS: Using a single kidney model 3 groups of 5 pigs each underwent 120, 150 and 180 minutes of warm ischemia, respectively. Microdialysis samples were collected before, during and after ischemia. Renal function assessments consisting of serum creatinine and GFR measurements were performed before ischemia and on post-ischemia days 1, 5, 9, 14 and 28. Kidneys exposed and not exposed to ischemia were collected for histological study. RESULTS: Interstitial glucose and pyruvate concentrations decreased, while lactate concentrations increased to stable levels during ischemia. Glutamate spiked at 30 minutes of ischemia and subsequently tapered, while glycerol increased throughout warm ischemia time. At post-ischemia day 28 renal function returned to pre-ischemia baseline levels in the group with 120 minutes of ischemia but did not recover to baseline in the 150 and 180-minute ischemic groups. Functional data correlated with histological findings. The 120-minute maximal renal tolerance of warm ischemia correlated with a mean +/- SD glycerol concentration of 167 +/- 24 micromol/l. CONCLUSIONS: Interstitial glycerol is a real-time, renal unit specific, minimally invasive marker of renal function deterioration. Exposure of porcine kidneys to ischemic insults resulting in renal cortical interstitial glycerol concentrations higher than 167 micromol/l is associated with irreparable functional damage in this model.

Effects of a preincisional 14-day course of valerian on natural killer cell activity in Sprague-Dawley male rats undergoing abdominal surgery.

INTRODUCTION: The American Cancer Society estimated that more than 1 million new cancer cases were diagnosed in 2005 and a majority of these patients died from metastatic spread. The standard for treating solid tumor cancer is surgical resection. However, it has been suggested that surgical resection may, in fact, promote metastasis. One of the body's natural defenses to combat metastasis is the activity of natural killer (NK) cells. NK cells serve as a vital mediator of detection during the early innate immune response and destruction of aberrant cells. It has been demonstrated that benzodiazepines may ameliorate surgery-induced suppression of NK cell activity. We examined the effect of a 14-day course of valerian, a herbal anxiolytic, on NK cell activity in Sprague-Dawley rats. METHODS: Thirty-five rats were assigned to 1 of 3 groups: (1) surgical animals administered research grade valerian, 15 mg/kg solubilized in peanut oil; (2) surgical animals administered peanut oil (vehicle); and (3) anesthesia-only animals administered valerian. One day before the 14-day course of valerian, blood was drawn to assay baseline NK cell activity. On experimental day, all animals were administered isoflurane anesthesia. Surgical animals underwent a standard laparotomy whereas anesthesia-only rats were anesthetized for the same period of time as the surgical rats. Twenty-four hours postexperiment animals underwent a second blood draw to assay NK cell activity. RESULTS: Analysis of covariance (ANCOVA) was used to analyze NK cell activity (measured in lytic units). Our results suggested that there was no difference (P = .9) in suppression within or between groups. CONCLUSIONS: Clinical studies with valerian have been published but with small numbers and some ambiguity. Further research regarding valerian's effectiveness as a modulator of NK cell activity and whether dosage or route of administration is a factor in modulation is still warranted.

Cyclic stretch attenuates effects of hyperoxia on cell proliferation and viability in human alveolar epithelial cells.

The treatment of severe lung disease often requires the use of high concentrations of oxygen coupled with the need for assisted ventilation, potentially exposing the pulmonary epithelium to both reactive oxygen species and nonphysiological cyclic stretch. Whereas prolonged hyperoxia is known to cause increased cell injury, cyclic stretch may result in either cell proliferation or injury depending on the pattern and degree of exposure to mechanical deformation. How hyperoxia and cyclic stretch interact to affect the pulmonary epithelium in vitro has not been previously investigated. This study was performed using human alveolar epithelial A549 cells to explore the combined effects of cyclic stretch and hyperoxia on cell proliferation and viability. Under room air conditions, cyclic stretch did not alter cell viability at any time point and increased cell number after 48 h compared with unstretched controls. After exposure to prolonged hyperoxia, cell number and [(3)H]thymidine incorporation markedly decreased, whereas evidence of oxidative stress and nonapoptotic cell death increased. The combination of cyclic stretch with hyperoxia significantly mitigated the negative effects of prolonged hyperoxia alone on measures of cell proliferation and viability. In addition, cyclic stretch resulted in decreased levels of oxidative stress over time in hyperoxia-exposed cells. Our results suggest that cyclic stretch, as applied in this study, can minimize the detrimental effects of hyperoxia on alveolar epithelial A549 cells.

Prolonged low-volume resuscitation with HBOC-201 in a large-animal survival model of controlled hemorrhage.

BACKGROUND: Military guidelines call for two 500-mL boluses of Hextend for resuscitation in far-forward environments. This study compared a hemoglobin-based oxygen carrier (HBOC-201; Hemopure) to Hextend when used to treat hemorrhagic shock in situations of delayed definitive care military operations. METHODS: Yorkshire swine (55-65 kg) were hemorrhaged to a mean arterial blood pressure (MAP) of 30 mmHg. Hypotension was maintained for 45 minutes followed by resuscitation with either Hextend (HEX) (n = 8) or HBOC-201 (HBOC) (n = 8). Over 8 hours, animals received up to 1,000 mL of either fluid in an effort to sustain an MAP of 60 mmHg. At the end of 8 hours, HEX animals received 2 L of lactated Ringer's solution followed by shed blood. HBOC animals received 4 L of lactated Ringer's solution only. Animals were killed and necropsied on postprocedure day 5. Hemodynamic data were collected during shock and resuscitation. Complete blood counts, amylase, lactate, coagulation studies, and renal and liver function were measured throughout the experiment. RESULTS: Equivalent volumes were hemorrhaged from each group (HBOC, 44.3 +/- 2.2 mL/kg; HEX, 47.4 +/- 3.0 mL/kg). The HBOC group achieved the goal MAP (HBOC, 60.0 +/- 2.3 mmHg; HEX, 46.4 +/- 2.3 mmHg; p < 0.01) and required less volume during the initial 8 hours (HBOC, 12.4 +/- 1.4 mL/kg; HEX, 17.3 +/- 0.3 mL/kg; p < 0.01). The HBOC group had lower SvO2 (HBOC, 46.3 +/- 2.4%; HEX, 50.7 +/- 2.5%; p = 0.12) and cardiac output (HBOC, 5.8 +/- 0.4 L/min; HEX, 7.2 +/- 0.6 L/min; p = 0.05), but higher systemic vascular resistance (HBOC, 821.4 +/- 110.7 dynes . s . cm-5; HEX, 489.6 +/- 40.6 dynes . s . cm-5; p = 0.01). Base excess, pH, lactate, and urine output did not differ between groups. HEX group survival was 50% (four of eight) versus 88% for the HBOC group (seven of eight). All animals survived the initial 8 hours. Animals surviving 5 days displayed no clinical or laboratory evidence of organ dysfunction in either group. CONCLUSION: HBOC-201 more effectively restored and maintained perfusion pressures with lower volumes, and allowed for improved survival. These data suggest that hemoglobin-based oxygen carriers are superior to the current standard of care for resuscitation in far-forward military operations.

The effect of the bovine hemoglobin oxygen therapeutic HBOC-201 on human neutrophil activation in vitro.

BACKGROUND: We previously demonstrated that HBOC-201 is an efficient resuscitation fluid. However, little is known about its immunomodulatory effects. The goal of this study was to investigate human neutrophil activation after exposure to HBOC-201 and other low-volume resuscitation fluids. METHODS: Whole blood from 10 healthy volunteers was serially diluted with HBOC-201, 7.5% hypertonic saline (HTS), 7.5% hypertonic saline-6% dextran 70 (HSD), Hextend, or PentaLyte. After an incubation period, samples were analyzed for oxidative burst and CD11b expression using flow cytometry. RESULTS: Increases in both CD11b and oxidative burst activity were noted in samples diluted with HBOC-201, Hextend, PentaLyte, and lactated Ringer's solution. Samples maximally diluted with HBOC-201 demonstrated increased CD11b expression when compared with lactated Ringer's solution and Hextend (196.11% vs. 22.68% and 33.97%; p = 0.018 and p = 0.033). CONCLUSION: Although differences were noted only at the highest dilutions, further studies investigating the effects of HBOC-201 are warranted.

The polymerized bovine hemoglobin-based oxygen-carrying solution (HBOC-201) is not toxic to neural cells in culture.

BACKGROUND: Recent data suggest that a neurotoxic effect of blood or its components may contribute to secondary neural cell dysfunction. This study investigated the effects of HBOC-201 (Hemopure) and purified human hemoglobin (hHgb) on rat fetal neural cell culture. METHODS: Neural cell cultures were exposed to HBOC-201 and hHgb (0.02, 0.2, 2.0, and 6.5 g/dL) for 24 hours, and then analyzed for proliferation, metabolism, and neurolysis. RESULTS: Cultures exposed to HBOC-201 maintained levels of proliferation and metabolism similar to controls while demonstrating no cellular lysis. However, cultures exposed to hHgb demonstrated decreased proliferation after exposure to 0.2, 2.0, and 6.5 g/dL hHgb (14,252.14, 3,221.89, and 343.12 vs. 19,509.53; p< 0.05) when compared with controls. In addition, cultures exposed to hHgb demonstrated decreased metabolic activity and increased cell lysis when compared with controls (p < 0.05). CONCLUSION: Cultures exposed to HBOC-201 displayed sustained metabolic activity and proliferation, and demonstrated no neurolysis, suggesting that HBOC-201 does not display the toxic characteristics of hHgb.