John R. Salsbury

Combined burn and smoke inhalation injury impairs ovine hypoxic pulmonary vasoconstriction

Objective:To examine the effects of combined burn and smoke inhalation injury on hypoxic pulmonary vasoconstriction, 3-nitrotyrosine formation, and respiratory function in adult sheep. Design:Prospective, placebo-controlled, randomized, single-blinded trial. Setting:University research laboratory. Subjects:Twelve chronically instrumented ewes. Interventions:Following a baseline measurement, sheep were randomly allocated to either healthy controls (sham) or the injury group, subjected to a 40%, third-degree body surface area burn and 48 breaths of cotton smoke according to an established protocol (n = 6 each). Hypoxic pulmonary vasoconstriction was assessed as changes in pulmonary arterial blood flow (corrected for changes in cardiac index) in response to left lung hypoxic challenges performed at baseline and at 24 and 48 hrs postinjury. Measurements and Main Results:Combined burn and smoke inhalation was associated with increased expression of inducible nitric oxide (NO) synthase, elevated NO2/NO3 (NOx) plasma levels (12 hrs, sham, 6.2 ± 0.6; injury, 16 ± 1.6 &mgr;mol·L−1; p < .01) and increased peroxynitrite formation, as indicated by augmented lung tissue 3-nitrotyrosine content (30 ± 3 vs. 216 ± 8 nM; p < .001). These biochemical changes occurred in parallel with pulmonary shunting, progressive decreases in Pao2/Fio2 ratio, and a loss of hypoxic pulmonary vasoconstriction (48 hrs, −90.5% vs. baseline; p < .001). Histopathology revealed pulmonary edema and airway obstruction as the morphologic correlates of the deterioration in gas exchange and the increases in airway pressures. Conclusions:This study provides evidence for a severe impairment of hypoxic pulmonary vasoconstriction following combined burn and smoke inhalation injury. In addition to airway obstruction, the loss of hypoxic pulmonary vasoconstriction may help to explain why blood gases are within physiologic ranges for a certain time postinjury and then suddenly deteriorate.

THE ATP-SENSITIVE POTASSIUM-CHANNEL INHIBITOR GLIBENCLAMIDE IMPROVES OUTCOME IN AN OVINE MODEL OF HEMORRHAGIC SHOCK

This study was designed as a prospective laboratory experiment to evaluate the effects of the ATP-sensitive potassium-channel inhibitor glibenclamide on hemodynamics and end-organ function in an ovine model of hemorrhagic shock. Twenty-four adult sheep were anesthetized and surgically prepared to measure hemodynamics of the systemic and pulmonary circulation. The anterior surface of the abdominal aorta was exposed at a location 6 cm superior to the iliac bifurcation. After a 60-min period of stabilization, this location was punctured with a 14-G needle. To induce a hemorrhagic hypotension (mean arterial pressure [MAP] less than 50 mmHg) via bleeding, the needle was left in place for 15 s to insure good blood flow. Thereafter, it was removed, and the abdomen closed. The animals were then randomized to receive either glibenclamide (4 mg/kg over 15 min) or an equal volume of the vehicle, started 1 h postinjury. Hemodynamic variables were measured every 30 min. Compared with the control group, MAP and systemic vascular resistance index (SVRI) were significantly higher in the intervention group throughout the entire 6-h study period. Ileal pH and urine output were higher in treated than in control animals (4 h, ileal pH 7.29 ± 0.31 vs. 7.17 ± 0.6; 6 h, urine output 36 ± 9 vs. 7.5 ± 2 mL; P value less than 0.05 each). Because glibenclamide improved both hemodynamics and organ function, it may be a beneficial component in the acute treatment of hemorrhagic shock.

Pulmonary vascular permeability changes in an ovine model of methicillin-resistant Staphylococcus aureus sepsis

IntroductionEndothelial dysfunction is a hallmark of sepsis, associated with lung transvascular fluid flux and pulmonary dysfunction in septic patients. We tested the hypothesis that methicillin-resistant Staphylococcus aureus (MRSA) sepsis following smoke inhalation increases pulmonary transvascular fluid flux via excessive nitric oxide (NO) production.MethodsEwes were chronically instrumented, and randomised into either a control or MRSA sepsis (MRSA and smoke inhalation) group.ResultsPulmonary function remained stable in the control group, whereas the MRSA sepsis group developed impaired gas exchange and significantly increased lung lymph flow, permeability index and bloodless wet-to-dry weight-ratio (W/D ratio). The plasma nitrate/nitrite (NOx) levels, lung inducible nitric oxide synthases (iNOS) and endothelial nitric oxide synthases (eNOS), vascular endothelial growth factor (VEGF) protein expressions and poly-(ADP)-ribose (PAR) were significantly increased by MRSA challenge.ConclusionsThese results provide evidence that excessive NO production may mediate pulmonary vascular hyperpermeability in MRSA sepsis via up regulation of reactive radicals and VEGF.

Ketorolac attenuates cardiopulmonary derangements in sheep with combined burn and smoke inhalation injury

Massive cutaneous burn combined with smoke inhalation causes high mortality in fire victims. Cyclo-oxygenase (COX) and inducible nitric oxide (NO) synthase (iNOS) have been shown to be up-regulated in burn injury. Ketorolac, a non-steroidal, anti-inflammatory agent (NSAID), inhibits prostaglandin and thromboxane synthesis through inhibition of COX. NSAIDs have been shown to down-regulate iNOS. Thus we hypothesized that treatment with ketorolac would attenuate burn/smoke-related cardiopulmonary derangements. We conducted a fully controlled long-term laboratory investigation in an Intensive Care Unit setting. Eighteen female sheep were surgically prepared for chronic study. After a recovery period of 5 days, a tracheotomy was performed under ketamine/halothane anaesthesia. Sheep were given a 40% total body surface third-degree burn and insufflated with cotton smoke (48 breaths, <40 degrees C). Sheep were divided into three groups: sham (not injured and not treated; n =6), control (injured, but not treated; n =6) and treated (injured and administered ketorolac 60 mg/day; n =6). The sham group had stable cardiopulmonary and systemic haemodynamics. Control animals showed depressed cardiopulmonary function, decreased pulmonary gas exchange, increased pulmonary microvascular leakage and decreased left ventricle stroke work index with elevated left atrial pressure. Systemic vascular leak in control animals was evidenced by robust haemoconcentration (haematocrit and fluid net balance). Treatment with ketorolac prevented all of these morbidities. Post-treatment with ketorolac also resulted in significant inhibition of elevated plasma nitrite/nitrate levels in control animals. These results suggest that ketorolac may ameliorate cardiopulmonary morbidity, at least in part, by inhibiting excessive NO.

Comparison of Gene Expression by Sheep and Human Blood Stimulated with the TLR4 Agonists Lipopolysaccharide and Monophosphoryl Lipid A

Background Animal models that mimic human biology are important for successful translation of basic science discoveries into the clinical practice. Recent studies in rodents have demonstrated the efficacy of TLR4 agonists as immunomodulators in models of infection. However, rodent models have been criticized for not mimicking important characteristics of the human immune response to microbial products. The goal of this study was to compare genomic responses of human and sheep blood to the TLR4 agonists lipopolysaccharide (LPS) and monophosphoryl lipid A (MPLA). Methods Venous blood, withdrawn from six healthy human adult volunteers (~ 28 years old) and six healthy adult female sheep (~3 years old), was mixed with 30 μL of PBS, LPS (1μg/mL) or MPLA (10μg/mL) and incubated at room temperature for 90 minutes on a rolling rocker. After incubation, 2.5 mL of blood was transferred to Paxgene Blood RNA tubes. Gene expression analysis was performed using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip. Agilent gene expression microarrays were scanned with a G2565 Microarray Scanner. Differentially expressed genes were identified. Results 11,431 human and 4,992 sheep probes were detected above background. Among them 1,029 human and 175 sheep genes were differentially expressed at a stringency of 1.5-fold change (p<0.05). Of the 175 sheep genes, 54 had a known human orthologue. Among those genes, 22 had > 1.5-fold changes in human samples. Genes of major inflammatory mediators, such as IL-1, IL-6 and IL-8, TNF alpha, NF-kappaB, ETS2, PTGS2, PTX3, CXCL16, KYNU, and CLEC4E were similarly (>2-fold) upregulated by LPS and MPLA in both species. Conclusion The genomic responses of peripheral blood to LPS and MPLA in sheep are quite similar to those observed in humans, supporting the use of the ovine model for translational studies that mimic human inflammatory diseases and the study of TLR-based immunomodulators.

Effect of hemorrhage rate on early hemodynamic responses in conscious sheep

Physiological compensatory mechanisms can mask the extent of hemorrhage in conscious mammals, which can be further complicated by individual tolerance and variations in hemorrhage onset and duration. We assessed the effect of hemorrhage rate on tolerance and early physiologic responses to hemorrhage in conscious sheep. Eight Merino ewes (37.4 ± 1.1 kg) were subjected to fast (1.25 mL/kg/min) and slow (0.25 mL/kg/min) hemorrhages separated by at least 3 days. Blood was withdrawn until a drop in mean arterial pressure (MAP) of >30 mmHg and returned at the end of the experiment. Continuous monitoring included MAP, central venous pressure, pulmonary artery pressure, pulse oximetry, and tissue oximetry. Cardiac output by thermodilution and arterial blood samples were also measured. The effects of fast versus slow hemorrhage rates were compared for total volume of blood removed and stoppage time (when MAP < 30 mmHg of baseline) and physiological responses during and after the hemorrhage. Estimated blood volume removed when MAP dropped 30 mmHg was 27.0 ± 4.2% (mean ± standard error) in the slow and 27.3 ± 3.2% in the fast hemorrhage (P = 0.47, paired t test between rates). Pressure and tissue oximetry responses were similar between hemorrhage rates. Heart rate increased at earlier levels of blood loss during the fast hemorrhage, but hemorrhage rate was not a significant factor for individual hemorrhage tolerance or hemodynamic responses. In 5/16 hemorrhages MAP stopping criteria was reached with <25% of blood volume removed. This study presents the physiological responses leading up to a significant drop in blood pressure in a large conscious animal model and how they are altered by the rate of hemorrhage.

Multivariate physiological recordings in an experimental hemorrhage model

In this paper we describe a data set of multivariate physiological measurements recorded from conscious sheep (N = 8; 37.4 ± 1.1 kg) during hemorrhage. Hemorrhage was experimentally induced in each animal by withdrawing blood from a femoral artery at two different rates (fast: 1.25 mL/kg/min; and slow: 0.25 mL/kg/min). Data, including physiological waveforms and continuous/intermittent measurements, were transformed to digital file formats (European Data Format [EDF] for waveforms and Comma-Separated Values [CSV] for continuous and intermittent measurements) as a comprehensive data set and stored and publicly shared here (Appendix A). The data set comprises experimental information (e.g., hemorrhage rate, animal weight, event times), physiological waveforms (arterial and central venous blood pressure, electrocardiogram), time-series records of non-invasive physiological measurements (SpO2, tissue oximetry), intermittent arterial and venous blood gas analyses (e.g., hemoglobin, lactate, SaO2, SvO2) and intermittent thermodilution cardiac output measurements. A detailed explanation of the hemodynamic and pulmonary changes during hemorrhage is available in a previous publication (Scully et al., 2016) [1].

Progression and variability of physiologic deterioration in an ovine model of lung infection sepsis

In this study, a lung infection model of pneumonia in sheep (n = 12) that included smoke inhalation injury followed by methicillin-resistant Staphylococcus aureus placement into the lungs was used to investigate hemodynamic and pulmonary dysfunctions during the course of sepsis progression. To assess the variability in disease progression, animals were retrospectively divided into survivor (n = 6) and nonsurvivor (n = 6) groups, and a range of physiological indexes reflecting hemodynamic and pulmonary function were estimated and compared to evaluate variability in dynamics underlying sepsis development. Blood pressure and heart rate variability analyses were performed to assess whether they discriminated between the survivor and nonsurvivor groups early on and after intervention. Results showed hemodynamic deterioration in both survivor and nonsurvivor animals during sepsis along with a severe oxygenation disruption (decreased peripheral oxygen saturation) in nonsurvivors separating them from survivor animals of this model. Variability analysis of beat-to-beat heart rate and blood pressure reflected physiologic deterioration during infection for all animals, but these analyses did not discriminate the nonsurvivor animals from survivor animals.NEW & NOTEWORTHY Variable pulmonary response to injury results in varying outcomes in a previously reported animal model of lung injury and methicillin-resistant Staphylococcus aureus-induced sepsis. Heart rate and blood pressure variability analyses were investigated to track the varying levels of physiologic deterioration but did not discriminate early nonsurvivors from survivors.

36: R-107 ATTENUATES SEVERITY OF ACUTE LUNG INJURY AFTER CHLORINE GAS INHALATION IN OVINE MODEL

Crit Care Med 2015 • Volume 43 • Number 12 (Suppl.) models to test the hypotheses that plasma products will suppress monocytes and that PF24 (plasma separated and frozen within 24 hr of collection) will suppress monocyte function more than FFP (plasma separated and frozen within 8 hr of collection). Methods: FFP and PF24 were obtained from the blood bank. 1 x 106 isolated monocytes from healthy adult donors were co-cultured with media plus 40% (by volume) of either plasma product or autologous plasma (control) for 18 hr at 37°C. This was followed by stimulation with LPS (1 ng/ml) for 4 hr. TNFα production was then quantified in supernatants by chemiluminescence. For doseresponse experiments, total plasma volume was kept at 40% but the ratio of FFP to autologous plasma was varied. Experiments were performed in at least 3 replicates, each with distinct monocyte donors and blood products. Data are mean ± SEM. Results: Exposure to FFP and PF24 both resulted in decreased LPS-induced TNFα production compared to autologous plasma controls (3146 ± 265 and 2829 ± 299 vs 6139 ± 1099 pg/ml; p = 0.01, RM-ANOVA, N=4). Differences between FFP and PF24 were not significant. In dose-response experiments, FFP exposure suppressed monocyte function with a linear dose-response relationship (%FFP:TNFα response (pg/ml): 0%: 8077 ± 1069, 10%: 5714 ± 515, 20%: 5068 ± 192, 30%: 3561 ± 39, 40%: 4135 ± 494; R2=0.65, p=0.0008, N=3). Conclusions: Plasma products suppress monocyte function in vitro. PF24 does not appear to be more immunosuppressive than FFP in our model. Further work is needed to identify mechanisms of plasma product-induced monocyte suppression, to evaluate other plasma products, and to translate these findings in clinical studies.