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Background: Logistics complicate fluid resuscitation of traumatic shock on the battlefield. Traumatic shock can result in oxygen debt (O(2)D) accumulation that is fatal. However, the ability of fluid strategies to repay O(2)D are not commonly reported. This pilot study examined various resuscitation fluids, including a combination of PEGylated bovine hemoglobin and hypertonic saline (AfterShock™) on their ability to repay O(2)D in traumatic shock.Methods: 41 anesthetized swine underwent hemorrhage to an O(2)D of 80 mL/kg. Animals received one of the following: 500 mL whole blood, 500 mL AfterShock™, 500 mL hypertonic (7.2%) saline, 250 mL hypertonic (7.2%) saline, 500 mL Hetastarch (6%), or 500 mL lactated Ringer's. Oxygen transport variables (O(2)D, oxygen consumption, oxygen delivery, central venous hemoglobin oxygen saturation, oxygen extraction ratios), lactate clearance, and survival were monitored for 3h after treatment. Data were analyzed using mixed-model ANOVA and comparisons were made to the performance of whole blood.Results: Only animals receiving AfterShock™, 500 mL hypertonic saline, and 500 mL Hetastarch survived to 180 min. While not statistically significant AfterShock™ demonstrated trends in improving the repayment of O(2)D and in improving oxygen transport variables despite having lower levels of global oxygen delivery compared to whole blood, Hetastarch and 500 mL hypertonic saline groups.Conclusion: Use of 500 mL AfterShock™, 500 mL of 7.2% saline or 500 mL of Hetastarch resulted in improved short-term survival. While not statistically significant, AfterShock™ demonstrated trends in improving O(2)D. These findings may have implications for designing resuscitation fluids for combat casualty care. [ABSTRACT FROM AUTHOR]

This issue of the ILAR Journal focuses on the topic of responsible science as it relates to animal research. We start with the concept of the scientist as a responsible citizen and then move through multiple phases of research including careful experimental planning, reporting, and incorporation of laboratory animal science. The work of the Institutional Animal Care and Use Committee (IACUC) or animal ethical/oversight body in reviewing both animal use and contributing to scientific excellence is explored. Additional topics include protection of animal handlers from multiple experimental hazards, use of agricultural animals and wildlife studies, regulatory ambiguities, and harmonization of animal research. Rounding out the issue is a discussion of how animal care and use programs can enhance animal welfare while mitigating regulatory burden, and our responsibility to clearly communicate the ethical use of animals in advancing biomedical research. A deeper understanding of these topics can assist scientists in simultaneously advancing their research and animal welfare., (© The Author(s) 2020. Published by Oxford University Press on behalf of the National Academies of Sciences, Engineering, and Medicine. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Biomedical research has made great strides in the past century leading to rapid advances in human life expectancy, all derived from improved understanding, prevention, and treatment of many diseases and conditions. Research involving laboratory animals has played a significant role in this medical progress. However, there continues to be controversy surrounding the use of animals in research, and animal models have been questioned regarding their relevance to human conditions. While research fraud and questionable research practices could potentially contribute to this problem, we argue that a relative ignorance of laboratory animal science has contributed to the "uncontrolled vivarium experiment" that runs parallel to the more controlled scientific experiment. Several variables are discussed, including husbandry, animal environment, social housing, and more, that can contribute to this uncontrolled experiment, and that can simultaneously decrease quality of life for rodent test subjects when ignored. An argument is put forward that laboratory animal veterinarians and scientists can and should play an important role in better controlling such variables. Similarly, the laboratory animal veterinarian and scientist should play an important role in responsible science by addressing complex interdisciplinary challenges., (© The Author(s) 2019. Published by Oxford University Press on behalf of the National Academy of Sciences. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The IACUC comprises the key component of animal research oversight at any institution or facility and thus has the responsibility to review and approve proposed animal activities. As the primary oversight unit that ensures the quality of animal welfare and therefore contributes to overall research quality, the IACUC can support reproducibility in research by ensuring rigorous experimental design, standardization of care and management, and assessment of the validity of research. An IACUC that is constituted as required by the Animal Welfare Act and the PHS Policy incorporates a wide range of expertise. Here we explore the contributions of the various IACUC members and discuss how each can help to enhance rigor and mitigate issues regarding irreproducibility in biomedical research involving animals.

Aims: Although there is evidence that deficits in bladder blood flow negatively impact bladder function, the effects of vesical, and perfusion pressures on bladder perfusion (perfusate flow), and of perfusate flow on vesical pressure, remain poorly understood. The present study used the isolated perfused working pig bladder model to examine the relationships between blood flow, and vesical and perfusion pressures., Methods: Vesical arteries of pig bladders obtained from a local slaughterhouse were cannulated and perfused with Krebs-Henseleit solution at different pressures, and with carbachol to cause bladder contraction. The urethra of each bladder was cannulated to permit filling (10 mL/min), isovolumetric contraction and emptying. A ureter was cannulated with a pressure sensor to monitor vesical pressure., Results: When at rest (50 mL vesical volume), bladder vesical pressure was 8.06 ± 1.5 mmHg and perfusate flow driven by a pressure gradient of 105 mmHg was 22.5 ± 2 mL/min (58.9 ± 7.8 mL/min-100 g). During filling, vesical pressure increased and flow decreased, but not necessarily in-parallel. Perfusate flow decreased transiently during isovolumetric contraction, and flow increased during emptying. A reduction in perfusion pressure from ∼105 to ∼40 mmHg reduced flow from ∼70 to ∼20 mL/min-100g, and reduced flow correlated with reduced vesical pressure., Conclusion: Perfusate flow is dependent on bladder perfusion pressure, and not necessarily reciprocally dependent on vesical pressure. Vesical pressure is highly sensitive to the level of perfusate flow, which supports the hypothesis that vesical pressure is dependent on the level of detrusor smooth muscle contractile activity (tone), and that compliance is dependent on bladder perfusion., (© 2017 Wiley Periodicals, Inc.)

Although recent studies reveal that activation of the metabolic and Ca 2+ sensor AMPK strongly inhibits smooth muscle contraction, there is a paucity of information about the potential linkage between pharmacological AMPK activation and vascular smooth muscle (VSM) contraction regulation. Our aim was to test the general hypothesis that the allosteric AMPK activator A-769662 causes VSM relaxation via inhibition of contractile protein activation, and to specifically determine which activation mechanism(s) is(are) affected. The ability of A-769662 to cause endothelium-independent relaxation of contractions induced by several contractile stimuli was examined in large and small musculocutaneous and visceral rabbit arteries. For comparison, the structurally dissimilar AMPK activators MET, SIM, and BBR were assessed. A-769662 displayed artery- and agonist-dependent differential inhibitory activities that depended on artery size and location. A-769662 did not increase AMPK-pT172 levels, but did increase phosphorylation of the downstream AMPK substrate, acetyl-CoA carboxylase (ACC). A-769662 did not inhibit basal phosphorylation levels of several contractile protein regulatory proteins, and did not alter the activation state of rhoA. A-769662 did not inhibit Ca 2+ - and GTPγS-induced contractions in β-escin-permeabilized muscle, suggesting that A-769662 must act by inhibiting Ca 2+ signaling. In intact artery, A-769662 immediately reduced basal intracellular free calcium ([Ca 2+ ] i ), inhibited a stimulus-induced increase in [Ca 2+ ] i , and inhibited a cyclopiazonic acid (CPA)-induced contraction. MET increased AMPK-pT172, and caused neither inhibition of contraction nor inhibition of [Ca 2+ ] i . Together, these data support the hypothesis that the differential inhibition of stimulus-induced arterial contractions by A-769662 was due to selective inhibition of a Ca 2+ mobilization pathway, possibly involving CPA-dependent Ca 2+ entry via an AMPK-independent pathway. That MET activated AMPK without causing arterial relaxation suggests that AMPK activation does not necessarily cause VSM relaxation.

Background: Many strategies have been utilized to treat traumatic shock via improved oxygen delivery (DO 2 ), while fewer have been used to in an attempt to reduce oxygen demand (VO 2 ). The cellular energy sensor 5' adenosine monophosphate-activated protein kinase (AMPK) has the potential to modulate both whole-body DO 2 and VO 2 . Therefore, we determined the effect of the AMPK activator AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribonucleoside) given acutely or chronically on key metabolites, hemodynamics, and oxygen consumption/delivery before and during hemorrhage in anesthetized male rabbits. Methods: Chronically treated animals received AICAR (40 mg/kg/day, IV) for 10 days prior to hemorrhage, while rabbits in the acute study were infused with AICAR (7.5 mg/kg bolus, 2 mg/kg/min infusion) or vehicle (0.3 ml/kg saline bolus, 0.03 ml/kg/min infusion) IV for 2 h prior to severe hemorrhage. Both acutely and chronically treated animals were sedated (ketamine/xylazine cocktail) the morning of the terminal experiment and surgically prepared for hemorrhage, including the implantation of arterial and venous catheters (for blood removal/sampling and drug/vehicle administration) and thoracotomy for implantation of transit-time flow transducers (for cardiac output determination). Results: AICAR given acutely lowered arterial blood glucose and increased blood lactate levels before hemorrhage, and abolished the well-documented hemorrhage-induced hyperglycemia seen in vehicle treated animals. Animals given AICAR chronically had blunted hemorrhage-induced hyperglycemia without prior baseline changes. Chronically treated AICAR animals showed significantly lower lactate levels during hemorrhage. Rabbits receiving AICAR both acutely and chronically experienced similar falls in mean arterial pressure, cardiac output and hence DO 2 to their vehicle counterparts throughout the hemorrhage period. However, rabbits treated either acutely or chronically with AICAR accumulated lower oxygen deficits and debt during hemorrhage compared to vehicle-infused controls. Conclusions: The oxygen debt data suggest that AMPK activation could decrease trauma associated morbidity and mortality, perhaps by mechanisms related to increased glucose utilization. Additional studies are needed to investigate the effects of AICAR and associated mechanisms of action when given during resuscitation from hemorrhage.

Metabolic stress diminishes smooth muscle contractile strength by a poorly defined mechanism. To test the hypothesis that metabolic stress activates a compensatory cell signaling program to reversibly downregulate contraction, arterial rings and bladder muscle strips in vitro were deprived of O 2 and glucose for 30 and 60 min ("starvation") to induce metabolic stress, and the phosphorylation status of proteins involved in regulation of contraction and metabolic stress were assessed in tissues under basal and stimulated conditions. A 15-30 min recovery period (O 2 and glucose repletion) tested whether changes induced by starvation were reversible. Starvation decreased basal phosphorylation of myosin regulatory light chain (MLC-pS19) and of the rho kinase (ROCK) downstream substrates cofilin (cofilin-pS3) and myosin phosphatase targeting subunit MYPT1 (MYPT1-pT696 and MYPT1-pT853), and abolished the ability of contractile stimuli to cause a strong, sustained contraction. Starvation increased basal phosphorylation of AMPK (AMPK-pT172) and 3 downstream AMPK substrates, acetyl-CoA carboxylase (ACC-pS79), rhoA (rhoA-pS188), and phospholamban (PLB-pS16). Increases in rhoA-pS188 and PLB-pS16 would be expected to inhibit contraction. Recovery restored basal AMPK-pT172 and MLC-pS19 to control levels, and restored contraction. In AMPKα 2 deficient mice (AMPK[Formula: see text]), the basal level of AMPK-pT172 was reduced by 50%, and MLC-pS19 was elevated by 50%, but AMPK[Formula: see text] did not prevent starvation-induced contraction inhibition nor enhance recovery from starvation. These results indicate that constitutive AMPK activity participates in constitutive regulation of contractile proteins, and suggest that AMPK activation is necessary, but may not be sufficient, to cause smooth muscle contraction inhibition during metabolic stress.

Overactive bladder is often characterized by biomechanical changes in the bladder wall, but there is no established method to measure these changes in vivo. The goal of this study was to develop a novel method to determine detrusor wall biomechanical parameters during urodynamics through the incorporation of transabdominal ultrasound imaging. Individuals with overactive bladder (OAB) underwent ultrasound imaging during filling. The fill rate was 10% of the cystometric capacity per minute as determined by an initial fill. Transabdominal ultrasound images were captured in the midsagittal and transverse planes at 1min intervals. Using image data and Pves, detrusor wall tension, stress, and compliance were calculated. From each cross-sectional image, luminal and wall areas along with inner perimeters were measured. In the sagittal and transverse planes, wall tension was calculated as Pves∗luminal area, wall stress as tension/wall area, and strain as the change in perimeter normalized to the perimeter at 10% capacity. Elastic modulus was calculated as stress/strain in the medial-lateral and cranial-caudal directions. Patient-reported fullness sensation was continuously recorded. Data from five individuals with OAB showed that detrusor wall tension, volume, and strain had the highest correlations to continuous bladder sensation of all quantities measured. This study demonstrates how detrusor wall tension, stress, strain, and elastic modulus can be quantified by adding ultrasound imaging to standard urodynamics. This technique may be useful in diagnosing and better understanding the biomechanics involved in OAB and other bladder disorders., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: Low amplitude rhythmic contractions (LARC) occur in detrusor smooth muscle and may play a role in storage disorders such as overactive bladder and detrusor overactivity. The purpose of this study was to determine whether LARC frequencies identified in vitro from strips of human urinary bladder tissue correlate with in vivo LARC frequencies, visualized as phasic intravesical pressure (p ves ) waves during urodynamics (UD)., Methods: After IRB approval, fresh strips of human urinary bladder were obtained from patients. LARC was recorded with tissue strips at low tension (<2 g) and analyzed by fast Fourier transform (FFT) to identify LARC signal frequencies. Blinded UD tracings were retrospectively reviewed for signs of LARC on the p ves tracing during filling and were analyzed via FFT., Results: Distinct LARC frequencies were identified in 100% of tissue strips (n = 9) obtained with a mean frequency of 1.97 ± 0.47 cycles/min (33 ± 8 mHz). Out of 100 consecutive UD studies reviewed, 35 visually displayed phasic p ves waves. In 12/35 (34%), real p ves signals were present that were independent of abdominal activity. Average UD LARC frequency was 2.34 ± 0.36 cycles/min (39 ± 6 mHz) which was similar to tissue LARC frequencies (p = 0.50). A majority (83%) of the UD cohort with LARC signals also demonstrated detrusor overactivity., Conclusions: During UD, a subset of patients displayed phasic p ves waves with a distinct rhythmic frequency similar to the in vitro LARC frequency quantified in human urinary bladder tissue strips. Further refinements of this technique may help identify subsets of individuals with LARC-mediated storage disorders.

Aims: The purpose of this investigation was to develop a non-invasive, objective, and unprompted method to characterize real-time bladder sensation., Methods: Volunteers with and without overactive bladder (OAB) were prospectively enrolled in a preliminary accelerated hydration study. Participants drank 2L Gatorade-G2® and recorded real-time sensation (0-100% scale) and standardized verbal sensory thresholds using a novel, touch-screen "sensation meter." 3D bladder ultrasound images were recorded throughout fillings for a subset of participants. Sensation data were recorded for two consecutive complete fill-void cycles., Results: Data from 14 normal and 12 OAB participants were obtained (ICIq-OAB-5a = 0 vs. ≥3). Filling duration decreased in fill2 compared to fill1, but volume did not significantly change. In normals, adjacent verbal sensory thresholds (within fill) showed no overlap, and identical thresholds (between fill) were similar, demonstrating effective differentiation between degrees of %bladder capacity. In OAB, within-fill overlaps and between-fill differences were identified. Real-time %capacity-sensation curves left shifted from fill1 to fill2 in normals, consistent with expected viscoelastic behavior, but unexpectedly right shifted in OAB. 3D ultrasound volume data showed that fill rates started slowly and ramped up with variable end points., Conclusions: This study establishes a non-invasive means to evaluate real-time bladder sensation using a two-fill accelerated hydration protocol and a sensation meter. Verbal thresholds were inconsistent in OAB, and the right shift in OAB %capacity-sensation curve suggests potential biomechanical and/or sensitization changes. This methodology could be used to gain valuable information on different forms of OAB in a completely non-invasive way., (© 2016 Wiley Periodicals, Inc.)

Aims: Previous studies using isolated strips of human detrusor muscle identified adjustable preload tension, a novel mechanism that acutely regulates detrusor wall tension. The purpose of this investigation was to develop a method to identify a correlate measure of adjustable preload tension during urodynamics., Methods: Patients reporting urgency most or all of the time based on ICIq-OAB survey scores were prospectively enrolled in an extended repeat fill-and-empty urodynamics study designed to identify a correlate of adjustable preload tension which we now call "dynamic elasticity." Cystometric capacity was determined during initial fill. Repeat fills to defined percentages of capacity with passive emptying (via syringe aspiration) were performed to strain soften the bladder. A complete fill with active voiding was included to determine whether human bladder exhibits reversible strain softening., Results: Five patients completed the extended urodynamics study. Intravesical pressure (p ves ) decreased with subsequent fills and was significantly lower during Fill 3 compared to Fill 1 (P = 0.008), demonstrating strain softening. Active voiding after Fill 3 caused strain softening reversal, with p ves in Fill 4 returning to the baseline measured during Fill 1 (P = 0.29). Dynamic elasticity, the urodynamic correlate of adjustable preload tension, was calculated as the amount of strain softening (or its reversal) per %capacity (Δaverage p ves between fills/Δ%capacity). Dynamic elasticity was lost via repeat passive filling and emptying (strain softening) and regained after active voiding regulated the process (strain softening reversal)., Conclusions: Improved understanding of dynamic elasticity in the human bladder could lead to both improved sub-typing and novel treatments of overactive bladder. Neurourol. Urodynam. 36:1086-1090, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Aims: The biomechanical properties of length adaptation and adjustable preload have been previously identified in detrusor smooth muscle in animal models. This in vitro study aims to show that human detrusor smooth muscle exhibits length adaptation and adjustable preload tension which could play an important role in both overactive bladder and detrusor underactivity., Methods: In order to demonstrate length adaptation, human detrusor smooth muscle strips are stretched and contracted beyond an optimum length and then contracted three times at the previous optimum length to determine if maximum active tension could be re-established. To demonstrate adjustable preload (Tap ), human detrusor smooth muscle strips are subjected to a pre-defined loading-unloading (strain softening) sequence to reduce preload. Then, tissues are contracted and the sequence is repeated to determine if this active process restored preload., Results: Nine patients (average age, 62) provide tissue: 89% are men with urothelial carcinoma and a minority (22%) also have neurogenic bladder dysfunction. In the length adaptation protocol, contractions show progressive increases in active tension (P < 0.05). In the Tap protocol, a significant amount of preload is lost to strain softening (P < 0.05) and is restored after active contraction (P = 0.50). Exposure to the rho-kinase inhibitor, H-1152, prevents the restoration of preload (P < 0.05)., Conclusions: This study demonstrates that human detrusor smooth muscle displays both length adaptation and Tap . Furthermore, Tap may be regulatable through a rho-kinase pathway. These biomechanical processes may be important in the pathophysiology of both overactive bladder and detrusor underactivity. Neurourol. Urodynam. 35:792-797, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

The decompensatory phase of hemorrhage (shock) is caused by a poorly defined phenomenon termed vascular hyporeactivity (VHR). VHR may reflect an acute in vivo imbalance in levels of contractile and relaxant stimuli favoring net vascular smooth muscle (VSM) relaxation. Alternatively, VHR may be caused by intrinsic VSM desensitization of contraction resulting from prior exposure to high levels of stimuli that temporarily adjusts cell signaling systems. Net relaxation, but not desensitization, would be expected to resolve rapidly in an artery segment removed from the in vivo shock environment and examined in vitro in a fresh solution. Our aim was to 1) induce shock in rabbits and apply an in vitro mechanical analysis on muscular arteries isolated pre- and postshock to determine whether VHR involves intrinsic VSM desensitization, and 2) identify whether net VSM relaxation induced by nitric oxide and cyclic nucleotide-dependent protein kinase activation in vitro can be sustained for some time after relaxant stimulus washout. The potencies of phenylephrine- and histamine-induced contractions in in vitro epigastric artery removed from rabbits posthemorrhage were decreased by ∼0.3 log units compared with the control contralateral epigastric artery removed prehemorrhage. Moreover, a decrease in KCl-induced tonic, relative to phasic, tension of in vitro mesenteric artery correlated with the degree of shock severity as assessed by rates of lactate and K(+) accumulation. VSM desensitization was also caused by tyramine in vivo and PE in vitro, but not by relaxant agents in vitro. Together, these results support the hypothesis that VHR during hemorrhagic decompensation involves contractile stimulus-induced long-lasting, intrinsic VSM desensitization., (Copyright © 2016 the American Physiological Society.)

Background: The ability to monitor the patient of hemorrhage noninvasively remains a challenge. We examined the ability of resonance Raman spectroscopy to monitor tissue hemoglobin oxygenation (RRS-StO2) during hemorrhage and compared its performance with conventional invasive mixed venous (SmvO2) and central venous (ScvO2) hemoglobin oxygen saturation as well as with near-infrared spectroscopy tissue hemoglobin oxygenation (NIRS-StO2)., Methods: Five male swine were anesthetized and instrumented followed by hemorrhage at a rate of 30 mL/min for 60 minutes. RRS-StO2 was continuously measured from the buccal mucosa, and NIRS-StO2 was continuously measured from the forelimb. Paired interval measures of SmvO2, ScvO2, and lactate were made. Pearson correlation was used to quantify the degree to which any two variables are related. Receiver operating characteristic (ROC) area under the curve values were used for pooled data for RRS-StO2, NIRS-StO2, SmvO2, and ScvO2 to compare performance in the ability of tissue oxygenation methods to predict the presence of an elevated arterial blood lactate level., Results: Sequential RRS-StO2 changes tracked changes in SmvO2 (r = 0.917; 95% confidence interval [CI], 0.867-0.949) and ScvO2 (r = 0.901; 95% CI, 0.828-0.944) during hemorrhage, while NIRS-StO2 failed to do so for SmvO2 (r = 0.283; 95% CI, 0.04919-0.4984) and ScvO2 (r = 0.142; 95% CI, -0.151 to 0.412). ROC curve performance of oxygenation measured to indicate lactate less than or greater than 3 mM yielded the following ROC area under the curve values: SmvO2 (1.0), ScvO2 (0.994), RRS-StO2 (0.972), and NIRS-StO2 (0.611)., Conclusion: RRS-StO2 seems to have significantly better ability to track central oxygenation measures during hemorrhage as well as to predict shock based on elevated lactate levels when compared with NIRS-StO2.

Exploratory pilot studies are crucial to best practice in research but are frequently conducted without a systematic method for maximizing the amount and quality of information obtained. We describe the use of response surface regression models and simultaneous optimization methods to develop a rat model of hemorrhagic shock in the context of chronic hypertension, a clinically relevant comorbidity. Response surface regression model was applied to determine optimal levels of two inputs--dietary NaCl concentration (0.49%, 4%, and 8%) and time on the diet (4, 6, 8 weeks)--to achieve clinically realistic and stable target measures of systolic blood pressure while simultaneously maximizing critical oxygen delivery (a measure of vulnerability to hemorrhagic shock) and body mass M. Simultaneous optimization of the three response variables was performed though a dimensionality reduction strategy involving calculation of a single aggregate measure, the "desirability" function. Optimal conditions for inducing systolic blood pressure of 208 mmHg, critical oxygen delivery of 4.03 mL/min, and M of 290 g were determined to be 4% [NaCl] for 5 weeks. Rats on the 8% diet did not survive past 7 weeks. Response surface regression model and simultaneous optimization method techniques are commonly used in process engineering but have found little application to date in animal pilot studies. These methods will ensure both the scientific and ethical integrity of experimental trials involving animals and provide powerful tools for the development of novel models of clinically interacting comorbidities with shock.

Knowledge of central venous pressure (CVP) is considered valuable in the assessment and treatment of various states of critical illness and injury. We tested a noninvasive means of determining CVP (NICVP) by monitoring upper arm blood flow changes in response to externally applied circumferential pressure to the upper arm veins. Thirty-six patients who were undergoing CVP monitoring as part of their care had NICVP determined and compared with CVP. Volume changes were measured in the upper arm using tetra-polar impedance plethysmography underneath a blood pressure cuff. The cuff was inflated over 5 s to a pressure greater than CVP but less than diastolic arterial pressure. After 45 to 60 s, the cuff was rapidly deflated. Noninvasive CVP was determined as the cuff pressure noted at the maximum derivative of the volume increase under the cuff during deflation. Noninvasive CVP was then compared with invasively measured CVP taken during the same period by Bland-Altman analysis. A total of 108 trials (three per subject) were performed on 36 patients. Mean bias was -0.26 mmHg (95% confidence interval [CI]: -0.67, 0.15). Limits of agreement were -2.7 and 2.2 mmHg with the 95% CI for the lower limit of agreement (-3.4, -2.0 mmHg) and for the upper limit of agreement (1.5, 2.9 mmHg). Correlation between CVP and NICVP was 0.95 (95% CI: 0.93 to 0.97; P < 0.0001). Noninvasive CVP as determined in this study may be a clinically useful substitute for traditional CVP measurement and may offer a tool for early diagnosis and treatment of acute states in which knowledge of CVP would be helpful.

Identification of occult shock is a major clinical problem compounded by inadequate criteria for assessing the efficacy of fluid resuscitation. We suggest that these problems may be resolved in part by understanding both the physiological mechanisms underlying oxygen debt accumulation and, more importantly, the debt repayment schedule during resuscitation. We present a simplified tutorial that incorporates the concept of the oxygen supply-delivery relationship with that of oxygen debt and show how this is relevant to the understanding of shock and resuscitation. Use of oxygen debt metrics as end points for shock have been controversial; however, much of the controversy may have been due to incomplete understanding of basic physiology of shock and semantic confusion between the various metrics proposed as end points. Here, we provide working definitions for the frequently misunderstood concepts of oxygen deficit and oxygen debt and discuss the relatively novel concept of oxygen debt repayment schedule. We introduce predictions made on the basis of data derived from animal models of hemorrhagic shock. Our calculations suggest that the amount of debt repaid in the first 2 h of resuscitation, rather than the restoration of volume per se, influences the likelihood of organ damage. Because of difficulties inherent in measuring oxygen debt in the prehospital and emergency settings, various metabolic end points such as lactate and base deficit have been proposed as surrogates. We demonstrate the heuristic value of this model in providing a predictive framework for both the optimum therapeutic time window and optimum fluid loadings before critical transitions to an irreversible shock state can occur. The model also provides an unambiguous and objective standard for quantifying the behavior of various postulated shock "markers".

Time profiles of arterial lactate concentrations have been proposed as markers for both the degree of physiological derangement during shock and effectiveness of clinical resuscitation, but have not been evaluated for use in short-term experimental protocols. We developed two quantitative mixed models of sequential arterial lactate concentrations to evaluate competing low-volume (<4 mL/kg) battlefield resuscitation therapies in a rat model of acute severe hemorrhagic shock: a simple linear additive model and a nonlinear mechanistic model that described lactate profiles in a continuous trajectory with a defined turning point. Data were obtained during a study evaluating a novel hemoglobin polymer (OxyVita) in a cocktail of hypertonic saline and Hextend as an alternative to standard Hextend. Fluids were either infused by titration to a mean systolic pressure of 60 mmHg or as a single bolus. Parameter estimates derived from both models were assessed for evidence of treatment efficacy and as indicators of short-term survival. A cocktail of hypertonic saline and Hextend was superior to standard Hextend in enhancing survival; however, lactate profiles did not differ between treatments. Regardless of resuscitation regimen, animals surviving to at least 60 min posthemorrhage can be discriminated from nonsurvivors by significantly lower peak lactates (a difference of at least 3 mM; P < 0.001), and all survivors exhibited a decline in lactate with resuscitation. Sequential measurements of lactate over relatively short time frames during resuscitation are of value in assessing both response to resuscitation and short-term mortality.

Background: Traumatic shock activates the hypothalamic-pituitary-adrenal axis (HPA) to mediate a cascade of defensive mechanisms that often include overwhelming inflammatory response and immunosuppression, which may lead to multiple organ failure. Androstenetriol (5 androstene, 3beta, 7beta, 17beta triol-AET) is a metabolite of dehydroepiandrosterone that markedly up regulates host immune response, prevents immune suppression, modulates inflammation and improves survival after lethal infections by pathogens and lethal radiation., Hypothesis: AET-induced immune modulation will improve survival in a conscious rodent model of traumatic shock., Methods: A relevant traumatic shock rodent model that applies to both combat and civilian sectors was used. After creation of a midline laparotomy (soft tissue trauma), animals were hemorrhaged to a mean arterial pressure of 35-40 mm Hg. Resuscitation was initiated sixty minutes later with crystalloid fluid and packed red blood cells and animals were observed for two days. In a randomized and blinded fashion, AET or vehicle was administered subcutaneously at the beginning of resuscitation., Results: In the vehicle group 5 out of 16 animals survived, (31%). In contrast, 9 out of 13 animals who received AET survived (69%), (Fisher Exact Test p < 0.05). Survival in the AET treatment group was associated with reduced levels of IL-6, IL-10, and IL-18, and enhanced IFN-gamma and IL-2 levels., Conclusion: : The results indicate that AET provides a significant protective effect and improves survival in a clinically relevant model of traumatic hemorrhagic shock. AET protective effects are associated with an elevation of Th1 and reduction of Th2 cytokines.

After severe hemorrhage, low-volume resuscitation with hypertonic fluids is increasingly preferred to more aggressive resuscitation strategies. Oxygen delivery to the tissues may be improved by augmentation with hemoglobin [Hb]-based oxygen-carrying compounds (HBOCs); however, previous studies have reported negative outcomes presumably related to extravasation of tetrameric Hb. The purpose of this study was to evaluate a novel large molecular weight polymer of cross-linked bovine Hb (OxyVita; OXYVITA Inc, New Windsor, NY) in a cocktail of hypertonic saline and Hextend (HX; HBOC-C) as an alternative to standard small-volume resuscitation using Hextend (HX) only. Outcomes were survival to 3 h and duration of MAP support more than 60 mmHg without additional fluid support. Conscious male Long-Evans rats were hemorrhaged to 60% total blood volume over 40 min. There were 4 groups: HBOC-C administered in a pressure-titrated infusion, HX titration, HBOC-C administered as a bolus, and HX bolus. Cardiovascular parameters, arterial gases, acid-base status, metabolites, electrolytes, Hb level, and oxygen saturation were measured at baseline, during each 20% hemorrhage increment, and 1, 2, and 3 h after the initiation of hemorrhage. Small-volume resuscitation with HBOC-C significantly improved survival to 3 h and improved MAP support times regardless of method of administration. However, physiological status at the end of hemorrhage significantly influenced survival regardless of resuscitation treatment. These results suggest that HBOC-augmented hypertonic cocktails are of promise in improving survival and providing target MAP support during small-volume resuscitation. Experimental evaluation of any resuscitation therapy should account for the degree of preexisting physiological compromise before therapy is initiated.

Pharmaceutically induced metabolic down-regulation may be a useful therapeutic adjunct when tissue oxygen supply is restricted. We hypothesized that protein synthesis inhibition in a non-hibernating species should lower oxygen demand, resulting in aerobic metabolic rate depression at the whole animal level. We compared metabolic responses and measures of systemic oxygenation of conscious catheterized rats given either protein synthesis inhibition (PSI) agents or carrier controls (normal saline and DMSO). Core temperature was measured by implanted transmitters, and VO2 was determined in an open flow-through metabolic chamber at 25 degrees C. Mean arterial pressure MAP and heart rate HR were determined from arterial pressure transducer tracings; arterial blood gases and lactate were sampled every 15 min. PSI rats exhibited an immediate transient decline in VO2, followed by a secondary decline to new resting levels; VO2 for the first hour was significantly lower than that for rats receiving DMSO vehicle. Unlike controls, PSI rats showed an overall 3.5 degrees C decline in core temperature, coupled with increased arterial lactate. There were no differences in MAP and HR of PSI rats compared to controls. Although hypothermic response to toxic agents typical of rodents cannot be ruled out completely, the mild hypothermia and reduced VO2 exhibited by PSI rats may be partially attributed to the action of protein synthesis agents.

Objective: This pilot study tested the potential of puromycin (PUR) to inhibit protein synthesis and reduce oxygen utilization in a non-hibernating, whole animal preparation., Methods: After anesthesia and instrumentation, male rats received a single dose of PUR or 0.9% saline (control), followed 60 min later with [(35)S] methionine/cysteine radiolabeling. Thirty minutes after isotope injection, organ biopsies were taken for quantification of de novo protein synthesis. Arterial and central venous blood gases were obtained at baseline and 60 min after injection of PUR or 0.9% saline. Temperature, mean arterial pressure (MAP), and heart rate were recorded continuously., Results: Animals receiving PUR demonstrated significant reductions in protein synthesis in all organ systems sampled (p<0.05). The overall reduction averaged 67.8%. Central venous oxygen saturations (S(cv)O(2)) were higher in the PUR group than the controls at 60 min (90+/-2% versus 80+/-4%, p<0.05). The oxygen extraction ratio (O(2)ER) decreased from 16.1+/-1.7% to 6.8+/-1.2% in the PUR group (p<0.05) and increased from 12.5+/-3.2% to 16.0+/-4.2% in the controls (p=0.44). There was no difference in temperature, MAP, heart rate or blood gas variables, other than S(cv)O(2), at baseline or 60 min between groups., Conclusions: These results demonstrate that PUR is capable of reducing whole body protein synthesis significantly within a relatively short duration of time. This appears to decrease whole body oxygen utilization as evidenced by an increase in S(cv)O(2) and a decrease in O(2)ER. Protein synthesis inhibition may reduce metabolic demands and should be tested for its potential to improve outcomes where oxygen demands exceed oxygen delivery.

Resonance Raman spectroscopy offers a mechanism for the noninvasive measurement of in vivo and in situ hemoglobin oxygen saturation (HbO(2)Sat) in living tissue. Clinically informative signals can be provided by resonance enhancement with deep violet excitation. It is notable that fluorescence does not significantly degrade the quality of the signals. During the controlled hemorrhage and resuscitation of rats, signal intensity ratios of oxy- vs. deoxyhemoglobin from sublingual mucosa correlated with co-oximetry values of blood withdrawn from a central venous catheter. The spectroscopic application described here has potential as a noninvasive method for the diagnosis of clinical shock and guidance of its therapy.

Intravenous perfluorocarbon emulsion (IV-PFC) has been shown to provide hemodynamic protection from gas embolism (Venous-VGE or arterial-AGE). The objective of this study was to investigate the mechanism of PFC protection from controlled VGE by quantifying the effects of IV-PFC emulsion on pulmonary elimination of nitrogen (N2). All rabbits received an intravenous pretreatment of PFC emulsion (Oxygent, 2.7 g/kg) or saline, then either a continuous room air infusion (0.25 ml/kg for 10 minutes) or a bolus of air (0.8 ml/kg within 10 seconds) through the femoral vein. Expiratory N2 peaked higher with PFC infusion immediately after air injection. The recovery to baseline of end tidal N2 was faster for PFC-treated animals (40 +/- 4.7 vs. 58 +/- 6.5 minutes). In PFC-treated animals, expired CO2, O2, arterial pressure and central venous pressure returned to baseline faster than the saline group. This study demonstrated that PFC increased pulmonary N2 washout. Correspondingly, PFC treatment better preserved the animals' hemodynamics after VGE injury. The use of IV-PFC promises to be a breakthrough non-recompression therapy for gas embolism in the treatment of Decompression Sickness (DCS) and in surgery.

Unlabelled: Trauma results in activation of the hypothalamic-pituitary-adrenal axis to mediate a cascade of neurohormonal changes as a defensive mechanism. Its prolongation, however, leads to a hypermetabolic, hypoperfused, and immunosuppressed state, setting the stage for subsequent sepsis and organ failure. Androstenetriol (5-androstene-3beta, 7beta, 17betatriol - AET), a metabolite of dehydroepiandrosterone, up-regulates the host immune response markedly, prevents immune suppression and controls inflammation, leading to improved survival after lethal infections by several diverse pathogens and lethal radiation. Such actions may be useful in improving survival from traumatic shock., Hypothesis: The neurosteroid AET will increase survival following traumatic shock., Methods: A combat relevant model of traumatic shock was used. Male Sprague-Dawley rats were anesthetized, catheterized and subjected to soft tissue injury (laparotomy). Animals were allowed to regain consciousness over the next 0.5 h and then bled 40% of their blood volume over 15 min. Forty-five minutes after the onset of hemorrhage animals were randomized to receive either a single subcutaneous dose of AET (40 mg/kg, sc) or vehicle (methylcellulose). Volume resuscitation consisted of l-lactated Ringer's (three times the shed blood volume), followed by packed red blood cells (one-third shed red cell volume). Animals were observed for three days., Results: A total of 24 animals were studied. Of the 12 animals randomized to receive AET, all (100%) survived compared to 9 of 12 animals (75%) randomized to receive the vehicle (p < 0.05)., Conclusion: AET significantly improved survival when administered subcutaneously in a single dose in this rodent model of traumatic shock. Further survival and mechanism studies are warranted.

Unlabelled: Knowledge of central venous pressure (CVP) is considered valuable in the assessment and treatment of various states of critical illness and injury., Objectives: We tested a noninvasive means of determining CVP (NICVP), by monitoring forearm volume changes in response to externally applied circumferential pressure to the upper arm veins., Methods: Sixteen patients who were undergoing CVP monitoring as a part of their care had NICVP determined and compared with CVP. Volume changes were measured in the forearm with mercury-in-silastic strain gauge plethysmography. A pressure cuff is placed in the upper extremity. The cuff is inflated over 5s to a pressure above CVP but below diastolic arterial pressure (40 mmHg). This allows blood into the forearm but prevents venous return. After 45-60 s the cuff is rapidly deflated. NICVP was determined as the cuff pressure noted at the maximum derivative of the forearm volume decrease during deflation. NICVP was then compared to invasively measured CVP taken during the same period., Results: A total of 48 trials (three per subject) were performed on 16 patients. The range of CVP recorded was 0-22 mmHg. The correlation between CVP and NICVP was 0.98 (95% CI: 0.95-0.98) (p<0.001). The bias between methods was 0.26 mmHg with the limits of agreement being 3.4 to -2.89 mmHg. When the average of three trials per patients was analysed the bias stayed at 0.26 mmHg but the limits of agreement improved to 2.54 and -2.03 mmHg., Conclusion: NICVP as determined in this study may be a clinically useful substitute for traditional CVP measurement and may offer a valid tool for early diagnosis and treatment of acute states in which knowledge of CVP would be helpful.

The purpose of this study was to correlate microvascular oxygen delivery (DO2) and consumption (VO2) in the skeletal muscle of spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKY) with hemodynamics during acute hypoxia. We expected greater abnormalities in central and microvascular hemodynamics during hypoxic induced shock in the SHR compared with the WKY due to microvascular rarefaction. The inspired oxygen fraction (FiO2) was lowered from 0.21 to 0.15, 0.1, 0.08, and 0.05 in anesthetized, mechanically ventilated rats. Lactate and base deficit values were similar for both groups at 0.21 and 0.15 FiO2, but higher in SHR at lower FiO2. Baseline aortic blood flow (SHR, 56.2+/-4.0 mL min; WKY, 61.8+/-5.3 mL min) and systemic DO2 (SHR, 9.02+/-0.82 mL min; WKY, 9.32+/-0.54 mL min) increased similarly when FiO2 was lowered to 0.15. Further reductions in FiO2 caused lower aortic flow and systemic DO2 in the SHR than WKY at 0.08 and 0.05 FiO2. Spinotrapezius blood flow increased from baseline (SHR, 24.8+/-1.8 nL s; WKY, 22.7+/-2.1 nL s) in both groups when FiO2 was reduced to 0.15; further reductions in FiO2 decreased blood flow in both groups, with lower values in the SHR group at 0.1 and 0.08 FiO2. The SHR group demonstrated higher venous oxygen saturation at low values of FiO2 compared with WKY. This reduced oxygen extraction in SHR resulted in a lower supply-dependent VO2 at low values of spinotrapezius DO2, perhaps attributed to arteriolar thickening and rarefaction seen in chronic hypertension.

Clinicians now realize the limitations of the physical examination in detecting compensated shock states, the severity of uncompensated states, and in determining the adequacy of resuscitation in order to prevent subsequent post-traumatic multisystem organ failure and death. A renewed interest has developed in interrogating the state of oxygen transport at the end-organ level in the trauma patient. Although used as a research tool and now clinically to monitor cerebral oxygenation during complex cardiovascular and neurosurgery, near infrared absorption spectroscopy (NIRS) is being more aggressively investigated and now marketed clinically as a noninvasive means to assess tissue oxygenation in the trauma patient at the end organ level. This paper will describe the principles of NIRS and the basis for its proposed use in the trauma patient to assess tissue oxygenation. This includes its known limitations, current controversies, and what will be needed in the future to make this technology a part of the initial and ongoing assessment of the trauma patient. The ultimate goal of such techniques is to prevent misassessment of patients and inadequate resuscitation, which are believed to be major initiators in the development of multisystem organ failure and death.

The purpose of this study was to develop a model of vascular injury in 8-week-old C3H/HeJ mice (weight, 25 to 30 g) by using air desiccation. The carotid arteries were excised 1 to 8 weeks postinjury and evaluated by Verhoeff's stain and immunocytochemistry. In the first group of mice studied (n = 107), neointimal formation occurred and peaked at Day 14. In addition, medial cell division (measured by bromodeoxyuridine labeling) peaked at Day 3, whereas intimal cell proliferation increased gradually throughout the experimental period of 21 days. In addition, extensive thrombus formation occurred within 3 days after injury. The next experiment involved 124 mice and evaluated the effect of anticoagulants on the neointimal and thrombotic response. Mice received aspirin, heparin, or vehicle-only time-release pellets. Both anticoagulants significantly decreased the neointimal and thrombotic responses. The results of this study validate our animal model as being consistent with the Response to Injury Hypothesis of atherogenesis.

We assessed the systemic effects of exchanges with blood or hemoglobin (Hb) raffimer under conditions of critical oxygen delivery (Do(2)crit). We compared Do(2)crit in animals receiving Hb-based oxygen carrier (HBOC; Hemolink), fresh blood (collected <24 h), or stored blood (10 days) before hemodilution. Rats were randomized to control, blood, or HBOC isovolemic exchange. Oxygen consumption was measured by using expired gas (o(2)a) and blood (o(2)b) samples, whereas whole-body oxygen delivery (Do(2)) was calculated from cardiac output and arterial oxygen content. After exchange, rats were subjected to stepwise isovolemic hemodilution. Blood pressure, gases, acid-base status, glucose, Hb oxygen saturation, heart rate, and total peripheral resistance were also measured. We found that 1) HBOC-treated rats showed an increased mean arterial blood pressure and total peripheral resistance throughout the hemodilution, 2) Do(2)crit calculated with o(2)a or o(2)b gave identical results, 3) Do(2)crit was not different between animals receiving blood and those receiving HBOC, 4) the terminal Hb concentration (1.8 +/- 0.1 g/dL) and Do(2) (5 +/- 1 mL . min(-1) . kg(-1)) were similar for all animals, and 5) most oxygen transport and biochemical variables changed similarly during hemodilution. The data suggest that tolerance to Do(2)crit is not altered by 50% replacement of native Hb by stored blood or Hb raffimer.

Systemic variables were evaluated with respect to O(2) delivery to test the hypothesis that critical O(2) delivery and critical Hb can be estimated by multiple variables collected simultaneously. Rats were subjected to transfusion with either fresh or stored blood and then subjected to stepwise isovolemic hemodilution. Critical levels were measured by the dual-regression method from plots of systemic variables against O(2) delivery and Hb. Delivery was calculated from cardiac index and arterial O(2) content. We found that 1) after hemodilution, O(2) delivery changed in a nonlinear relationship with Hb; 2) critical delivery calculated using 30 different systemic variables was not statistically different from each other; 3) critical delivery and critical Hb were correlated but were not different between animals receiving fresh or stored blood; and 4) similar critical levels were found using a single variable from several animals and using several variables from the same subject. The best variables to estimate critical delivery were lactate, bicarbonate, base excess, O(2) extraction ratio, expired CO(2), pulse pressure, cardiac index, and systolic pressure. The data suggest that a multivariable analysis of critical delivery may help determine the physiological oxygenation boundary at the whole body level. This may assist in finding therapeutic triggers on an individual basis using systemic markers of the transition from aerobic to anaerobic metabolism.

We tested the hypotheses that continuous total peripheral resistance (TPR) measurements are superior to intermittent data collection and that variables related to TPR can be used to distinguish between survivors and nonsurvivors (NS), respectively, of prolonged hemorrhagic hypotension (HH). One week after a transit-time ultrasound probe was implanted on their ascending aortas, 21 rats were subjected to 4 h of HH at 40 mmHg. Measurements were made before and up to 4 h after initiation of HH. Additional bleeding or Ringer L-lactate (RL) infusion was used to maintain HH. TPR was continuously measured online using recordings of blood flow and arterial pressure. Approximately 67% of the rats survived > or =3 h; others were considered NS. Data collected at 30-min intervals failed to detect the maximum value of TPR (TPRmax). The times to reach TPRmax were similar for survivors and NS and were strongly correlated with the bleeding end points and with the RL infusion-onset times. However, survivors showed higher TPRmax values than NS (P <0.005) and had a significantly longer period than NS during which TPR was above baseline level (116 +/- 20 vs. 51 +/- 10 min). In conclusion, 1) the transit-time ultrasound technique at high sampling rate allowed continuous and accurate real-time monitoring of TPR, 2) the bleeding end point and RL infusion-onset times may be used as surrogates of the time to TPRmax, 3) TPRmax of survivors and NS could be detected only using a continuous TPR measurement, and 4) differences between survivors and NS could be revealed by the continuous TPR curve.

This study describes the implantation of transit-time flow probes on the ascending aorta of rats while minimizing the risk of postoperative complications. Special emphasis is placed on our new method of rat intubation as well as the production of materials necessary for the implantation procedure such as endotracheal tubes and heparin bonded vessel catheters. The effects of these devices on the response to acute hypoxia were studied in rats following a 5-7 day recovery from the implantation procedure. Systemic and microvascular measurements were made on instrumented rats (n = 5) and non-instrumented controls (n = 3) that were ventilated with 21%, 15%, 10%, 8% and 5% oxygen. Arterial pressure, PO(2), lactate, and base deficit were not different between the implanted and control animals at any inspired oxygen concentration. Microvascular flow in the primary arterioles of the spinotrapezius muscle was also similar between the two groups at all inspired oxygen concentrations. We conclude that this novel methodology facilitates the measurement of whole body oxygen delivery in resting and haemodynamically-stressed rats.

We tested the hypothesis that a deficit in oxygen extraction or an increase in oxygen demand after skeletal muscle contraction leads to delayed recovery of tissue oxygen tension (Po(2)) in the skeletal muscle of hypertensive rats compared with normotensive rats. Blood flow and Po(2) recovery at various sites in the spinotrapezius muscle of spontaneously hypertensive rats (SHRs) were evaluated after a 3-min period of muscle contraction and were compared with corresponding values in Wistar-Kyoto rats (WKYs). The recovery of tissue Po(2) [75 +/- 7 (SHRs) vs. 99 +/- 12% (WKYs) of resting values] and venular Po(2) [72 +/- 13 (SHRs) vs. 104 +/- 10% (WKYs) of resting values] were significantly depressed in the SHRs 30 s postcontraction. The delayed recovery persisted for 120 s postcontraction for both tissue [86 +/- 11 (SHRs) vs. 119 +/- 13% (WKYs) of resting values] and venular [74 +/- 2 (SHRs) vs. 100 +/- 9% (WKYs) of resting values] Po(2) levels. There was no significant difference in the recovery of arteriolar Po(2) between the two groups 30 s postcontraction [95 +/- 7 (SHRs) vs. 84 +/- 8% (WKYs) of resting values]. Values for resting diameter of arcade arterioles in the two groups were not different [52 +/- 3 (SHRs) vs. 51 +/- 3 microm (WKYs)], but the arteriolar diameter after the 3-min contraction period was greater in the SHRs (71 +/- 4 microm) than the WKYs (66 +/- 4). Likewise, red blood cell (RBC) velocity [5.8 +/- 0.3 (SHRs) vs. 4.7 +/- 0.2 mm/s (WKYs)] and blood flow [23.0 +/- 0.8 (SHRs) vs. 16.0 +/- 1.0 nl/s (WKYs)] measurements were significantly greater in the SHRs at 30 s postcontraction. The delayed recovery of tissue Po(2) in the SHRs compared with the WKYs can be explained by a decrease in oxygen diffusion from the rarefied microvascular network due to the increased RBC velocity and the shorter residence time in the microcirculation and the consequent disequilibrium for oxygen between plasma and RBCs. The delayed recovery of venular Po(2) in the SHRs is consistent with this explanation, as venular Po(2) is slowly restored to baseline by release of oxygen from the RBCs. This leaves the arterioles in the primary role as oxygen suppliers to restore Po(2) in the tissue after muscle contraction.

Studies are needed to provide a rigorous examination of the relevance of monitored variables during prolonged hemorrhagic hypotension (HH). This study was designed to investigate the parameters that describe biochemical and O2 transport patterns in animals subjected to HH. Systemic parameters that could differentiate survivors from nonsurvivors were identified. An aortic flow probe was implanted in rats (n = 21) for continuous measurement of cardiac output. Experiments were performed 6-9 days after surgery. Rats were bled to a mean arterial pressure of 40 mmHg and kept at that level using Ringer-lactate solution. Arterial and venous blood pressures, gases, acid-base status, glucose, lactate, electrolytes, hemoglobin, O2 saturation, heart and respiratory rates, total peripheral resistance, and O2 delivery and consumption were measured before hemorrhage, soon after 40 mmHg was reached, and 0.5, 1, 2, 3, and 4 h later. Fifty-three percent of rats survived > or =3 h (survivors); others were considered nonsurvivors. Nonsurvivors showed a significantly greater degree of metabolic acidosis than survivors. Arterial PO2, respiratory rate, O2 saturation, O2 content, glucose, and pH were significantly higher in survivors. The rate of Ringer-lactate infusion, arterial K+, and PCO2 were lower in survivors. Arterial K+ and respiratory rate were the only parameters significantly different between survivors and nonsurvivors at all time points during HH. Arterial levels of K+ showed the clearest distinction between survivors and nonsurvivors and may explain the sudden death experienced by animals during HH. The data suggest that early respiratory and metabolic compensations are essential for survival of prolonged HH.

Thrombotic thrombocytopenic purpura (TTP) is a rare clinical entity. It is a multi-systemic disorder characterized by a clinical pentad of thrombocytopenia, microangiopathic hemolytic anemia, diffuse and nonfocal neurologic symptoms, decreased renal function, and fever. Abdominal pain is an uncommon presenting symptom for TTP. Pancreatitis may occur from TTP or, in a few cases, may trigger TTP. The clinical diagnosis of TTP is generally difficult because there are many varied clinical presentations and the full expression of the pentad may be prolonged. However, once the diagnosis is suspected or confirmed, immediate plasmapherseis with plasma exchange must be performed to reduce the severe morbidity from neurologic disability.

This study tested the hypothesis that removal of fatty acids as a fuel source would improve cardiac efficiency at the expense of reduced cardiac contractile function in the isolated working heart after hemorrhage-retransfusion. Non-heparinized male Sprague-Dawley rats were anesthetized with ketamine-xylazine and were hemorrhaged to a mean arterial blood pressure of 40 mmHg for 1 h. Two-thirds volume of shed blood was reinfused together with 0.9% NaCl in a volume equal to 2.3 times the shed blood volume, followed by continuous infusion of 0.9% NaCl at 10 mL/kg per h for 3 h. Hearts were removed and perfused in closed, recirculating working mode for 60 min to measure hydraulic work and cardiac efficiency. Rates of glycolysis and glucose oxidation were assessed with [5-3H/U-14C] glucose (11 mM) in the absence or presence of 0.4 mM palmitate. Compared to baseline measurements, hemorrhage-retransfusion significantly reduced arterial blood glucose (228+/-7 versus 118+/-12 mg/dL) and non-esterified fatty acid concentrations (0.36+/-0.01 versus 0.30+/-0.02 mM), while elevating blood lactate (0.8+/-0.1 versus 2.5+/-0.4 mM). Perfusion of sham hearts with glucose-only did not alter cardiac work compared to shams perfused with glucose plus palmitate. However, shocked hearts perfused with glucose-only demonstrated a significant reduction in cardiac work compared to shocked hearts perfused with glucose plus palmitate and compared to sham hearts perfused with glucose only (P < 0.05, repeated measures ANOVA). Shocked hearts perfused with glucose plus palmitate showed no reduction in cardiac work compared to shams. Shocked hearts perfused with glucose-only had increased glucose oxidation rates compared to shams perfused with glucose plus palmitate. In sham hearts perfused with glucose-only, myocardial glycogen and triacylglycerol contents were significantly reduced compared to hearts freeze-clamped in situ. These endogenous fuels were not decreased in shocked hearts. These data indicate that hemorrhagic shock renders the heart unable to mobilize endogenous fuels, and suggest that withdrawal of fatty acid oxidation will impair myocardial energy metabolism during resuscitation.

This study was undertaken to examine the role of lactate on cardiac function and metabolism after severe acute hemorrhagic shock. Anesthetized, nonheparinized rats were bled to a mean arterial pressure of 25-30 mm Hg for 1 h; controls were not bled. Their hearts were removed, and cardiac work and efficiency (work/oxygen consumption) were measured in the isolated working heart mode for 60 min. The hearts were perfused with one of five substrate combinations: 1) glucose (11 mM), 2) glucose + 0.4 mM palmitate, 3) glucose + 0.4 mM palmitate + 8.0 mM lactate, 4) glucose + 1.2 mM palmitate, or 5) glucose + 1.2 mM palmitate + 8.0 mM lactate. After perfusion, hearts were freeze-clamped, and tissue contents of free coenzyme-A (CoA), acetyl CoA, and succinyl CoA were measured, as was myocardial pyruvate dehydrogenase (PDH) activity. The addition of 8.0 mM lactate significantly improved cardiac work in shocked hearts perfused with 0.4 mM palmitate and increased cardiac efficiency in the presence of either 0.4 mM or 1.2 mM palmitate. Compared to control hearts, shocked hearts exhibited a 20-30% decrease in PDH activity. Shocked hearts perfused with lactate demonstrated no increase in acetyl CoA content but did have a significant increase in tissue succinyl CoA compared to control hearts perfused with lactate or shocked hearts perfused without lactate. In the heart recovering from severe hemorrhagic shock, lactate improves cardiac efficiency in the presence of free fatty acids, possibly by a anaplerosis of the tricarboxylic acid cycle.

We have demonstrated previously that dichloroacetate (DCA) treatment in rodents ameliorates, via activation of the pyruvate dehydrogenase complex, the cardiovascular depression observed after hemorrhagic shock. To explore the mechanism of this effect, we administered DCA in a large animal model of hemorrhagic shock. Mongrel hounds were anesthetized with 1.5% isoflurane and were measured for hemodynamics, myocardial contractility, and myocardial substrate utilization. They were hemorrhaged to a mean arterial pressure of 35 mm Hg for 90 min or until arterial lactate levels reached 7.0 mM (1137 +/- 47 mL or 49 +/- 2% total blood volume). Animals were chosen at random to receive DCA dissolved in water or an equal volume of saline at the onset of resuscitation. Two-thirds of the shed blood volume was returned immediately after giving an equivalent volume of saline. Two hours after the onset of resuscitation, mean arterial pressure was not different between DCA and control groups (79 +/- 3 vs. 82 +/- 3 mm Hg, respectively). Arterial lactate levels were significantly reduced by DCA (0.5 +/- 0.06 vs. 2.0 +/- 0.2 mM). However, DCA treatment was associated with a decreased stroke volume index (0.56 +/- 0.06 vs. 0.82 +/- 0.08 mL/kg/beat) and a decreased myocardial efficiency (19 vs. 41 L x mm Hg/mL/100 g tissue). During resuscitation by DCA, myocardial lactate consumption was reduced (21.4 +/- 3.7 vs. 70.7 +/- 16.3 micromole/min/100 g tissue) despite a three-fold increase in myocardial pyruvate dehydrogenase activity, while free fatty acid levels actually began to rise. Although increased lactate oxidation should be beneficial during resuscitation, we propose that DCA treatment led to a deprivation of myocardial lactate supply, which reduced net myocardial lactate oxidation, thus compromising myocardial function during resuscitation from hemorrhagic shock.

Objective: Test whether brief deep hemorrhagic hypotension or prolonged moderate hemorrhagic hypotension impairs intrinsic heart function., Methods: Pentobarbital-anesthetized, non-anticoagulated rats were cannulated via the carotid artery. This study focuses on three main groups: 1) hemorrhage to a mean arterial blood pressure (MAP)=25 mm Hg for 1 h (1 h severe shock), 2) hemorrhage to MAP=40 mm Hg for 3 h (3 h moderate shock), 3) no hemorrhage (control). Hearts were either freeze-clamped in-situ for tissue analysis (n=6 per group) or were removed to study in vitro cardiac function and efficiency using a working heart perfusion (n=12 per group, glucose (11 mM)/palmitate (0.4 mM), 3% BSA buffer). Following perfusion, hearts were freeze-clamped and analyzed for free CoA, acetyl-, succinyl-, and malonyl-CoA, ATP content and for TNF-alpha content., Results: Isolated working hearts obtained following 1 h of severe shock generated 20% less hydraulic work than hearts obtained from control rats or rats subjected to 3 h of moderate shock. The cardiac efficiency (work/O2 consumption) was also significantly reduced with 1 h severe shock (0.76 +/- 0.07 after 15 min perfusion) versus control (0.96 +/- 0.06) or 3 h prolonged shock (1.10 +/- 0.09). Myocardial Co-A ester, ATP and TNF-alpha concentrations were not different between control and shocked hearts, although TNF-alpha concentrations increased significantly in all hearts during ex vivo perfusion., Conclusions: Depth of hypotension is more important than duration in causing intrinsic cardiac dysfunction. This post-hemorrhagic cardiac dysfunction is not a result of substrate limitation to the heart, nor myocardial TNF-alpha accumulation, but is more likely a result of impaired transfer of energy from molecular oxygen into external cardiac work.

Resuscitation with crystalloid and packed red blood cells has for the most part replaced the use of plasma and whole blood in the initial treatment of hemorrhagic shock. The effects of such changes on cardiovascular function following hemorrhagic shock remain largely unexplored. We examined cardiovascular function in anesthetized canines subjected to severe hemorrhagic shock. Mongrel canines of either gender were anesthetized with isoflurane and instrumented for measurement of arterial pressure, cardiac output, coronary flow, and left ventricular pressure and volume for the determination of end systolic elastance (Ees). Following a 30-min stabilization period, blood was rapidly removed to induce fixed pressure (mean arterial pressure = 35 mmHg) hemorrhagic shock for 90 min or until an arterial lactate of 7.0 mM was achieved. Animals were then resuscitated with 2/3 of the shed volume as lactated Ringer's and an equal volume of either whole blood (WB, n = 8) or packed red blood cells (PRBC, n = 10) resuspended in lactated Ringer's (LR) solution to replace expressed plasma volume. PRBC resuscitated dogs showed lower values of mean arterial pressure, cardiac output, rates of ventricular contraction and relaxation and myocardial work. Increasing the maintenance infusion rate of LR (10 mL/kg/h) following PRBC infusion normalized mean arterial pressure, but not other indices of cardiovascular function. Thus, WB, but not PRBC resuscitation restores normal myocardial function during resuscitation from severe hemorrhagic shock.

Hyporeactivity of vessels to constrictor agents is thought to contribute to cardiovascular decompensation following trauma-hemorrhage and resuscitation. In this study, we determined if inhibition of poly(ADP-ribose) synthetase (PARS) activity prevented the development of vascular hyporeactivity in rats following trauma-hemorrhage and resuscitation. Trauma consisted of a laparotomy that was closed and rats were hemorrhaged into a reservoir containing citrate to 40 mm Hg for 90 min. Resuscitation included 2/3 of the shed blood plus 2 1/3 of the shed volume as Ringer's lactate. Sham animals received the laparotomy and were time-matched. Induction of iNOS was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Aortic rings isolated 6 h after the initiation of hemorrhage (4.5 h after resuscitation) showed decreased responsiveness to norepinephrine (peak developed tension 0.31+/-0.01 g/mg tissue) compared with sham rings (0.43+/-0.02 g/mg tissue), but no change in EC50 for this response (approximately 5x10(-8) M). Addition of the PARS inhibitor, 3-aminobenzamide, at the onset of resuscitation prevented the decrease in response of aortic rings. The addition of the structural analogue, 3-aminobenzoic acid, which does not inhibit PARS, did not prevent the decrease in vascular reactivity. These agents did not alter vascular responses to norepinephrine in sham animals. iNOS induction was not associated with depressed contractile function. These results indicate that decreased vascular reactivity was prevented by inhibition of PARS and that PARS activation was independent of iNOS induction following trauma-hemorrhage and resuscitation.

Background and Purpose: Standard treatment for massive hemorrhage in dogs is infusion of whole blood or of packed red blood cells with fresh frozen plasma if whole blood is not available. Although most whole blood is collected using a citrate-based anticoagulant, knowledge of citrate's relevant non-anticoagulant effects is not widespread. Citrate's anticoagulant activity is achieved through chelation of divalent metal cations (e.g., magnesium, calcium), which may exacerbate cardiovascular and metabolic insults attributable to hemorrhage., Methods: Blood pressures, gas tensions, metabolites, and electrolytes; myocardial metabolites, pressures, and contractility; cardiac output; and left cranial descending and circumflex coronary artery flows were measured in 21 anesthetized dogs after hemorrhage was induced by collection of blood into a citrated reservoir to mean arterial pressure of 45 mm Hg for approximately 60 min (until arterial lactate concentration was 7.0 mmol/L), followed by a 1-h transfusion and 2 h of maintenance., Results: Arterial ionized calcium concentration, total peripheral resistance, and myocardial function decreased significantly during hemorrhage. All aforementioned responses but myocardial function continued to decrease during the initial 20 min of transfusion, then began to recover. Total peripheral resistance and end-systolic elastance were the only factors significantly related to calcium concentration., Conclusion: Transfusion with citrated whole blood may significantly alter calcium concentration, negatively affecting myocardial and vascular function.

Objectives: This study tested whether the combination of dobutamine echocardiography (DE) and myocardial contrast echocardiography (MCE) was superior to either technique alone in identifying postischemic myocardium and in differentiating it from necrotic myocardium., Background: Wall motion abnormalities at rest occur in postischemic myocardium in the presence of infarction, stunning or hibernation, alone or in combination. Various investigators have suggested that either DE or MCE can be used to identify the presence of myocardial viability., Methods: We studied a total of 53 mongrel dogs in an open chest model of coronary occlusion of various durations followed by reperfusion and dobutamine administration (10 microg/kg body weight per min). MCE with aortic root injections of Albunex (area under the curve) and DE (percent thickening fraction) were performed at the different stages. Postmortem triphenyltetrazolium chloride (TTC) staining was used to identify myocardial necrosis., Results: Thirteen dogs underwent brief (15 min) occlusions and developed no necrosis (Group I). Of 40 dogs that underwent prolonged (30 to 360 min) occlusions, 14 had no infarction (Group II), whereas 26 did (Group III: 12 papillary muscle, 7 subendocardial, 7 transmural). MCE (expressed as percent change from baseline) demonstrated changes that paralleled the blood flow changes observed by radiolabeled microspheres at all interventions (r = 0.67, p < 0.0001). Regional ventricular function improved with dobutamine administration in the ischemic region in all three groups. The sensitivity (88%) for detecting myocardial viability was superior when the two techniques were combined; however, a poor specificity (61%) was observed., Conclusions: Contractile reserve and perfusion data are complementary when assessing regional wall motion abnormalities in postischemic myocardium. DE alone cannot differentiate postischemic from infarcted myocardium; simultaneous data on myocardial perfusion are required. The combination of DE and MCE is superior to either technique alone for identifying the absence of myocardial necrosis.

During myocardial infarction, lack of myocardial opacification after reperfusion has been associated with poor or no recovery of function. We have previously documented the presence of perfusion abnormalities after brief coronary occlusions without infarction and the absence of perfusion abnormalities after prolonged occlusions with infarction. To characterize myocardial perfusion patterns immediately after reperfusion, we studied 53 animals in two groups in a coronary occlusion-reperfusion model. Temporary occlusions (group 1, 15 minutes; group 2, 30 to 360 minutes) were performed, followed by reperfusion with and without dobutamine. Myocardial contrast echocardiography was performed with aortic root injections of sonicated 5% serum human albumin (Albunex) during each intervention. Group 1 dogs showed no evidence of myocardial infarction. In group 2, 26 of 40 dogs had infarctions. After reperfusion, no perfusion abnormalities were seen in 13 of 26 group 2 dogs with infarctions; perfusion abnormalities were identified after reperfusion in 2 of 13 group 1 and in 8 of 14 group 2 dogs without infarctions. In animals subjected to prolonged ischemia, the absence of perfusion abnormalities after reperfusion did not rule out the presence of necrosis. Similarly, in animals without infarction subjected to ischemia, the presence of a perfusion defect after reperfusion did not represent the presence of necrosis but an abnormal microvascular reserve. These results suggest that early after reperfusion, assessment of perfusion by myocardial contrast echocardiography has significant limitations in the evaluation of myocardial viability and salvage.

Background: The long-term success of heart transplantation continues to be in jeopardy because of the development of accelerated vascular myointimal proliferation. Transfer of genes encoding products that can modulate the adverse consequences of phenomena that cause myointimal proliferation, into the allograft vessel wall, may modify these pathologic processes. The purpose of this study was to assess the feasibility of gene transfer and to evaluate the duration of gene expression in a rabbit heterotopic aortic transplant model of allograft vasculopathy., Methods: The abdominal aortas of 32 outbred New Zealand rabbits were harvested and cross-sectionally bisected (n = 64). Six donor and recipient animals were used in a preliminary study to examine neointimal proliferation without accompanying gene transfer. Of the remaining 26 rabbits (52 allografts), one half of each allograft aorta was administered a control solution, while the other half was incubated with a replication-defective, recombinant, adenoviral vector-encoding, cytomegalovirus promoter-regulated beta-galactosidase. After a 20-minute incubation period, bilateral aorto-carotid transplantations were performed in 26 recipient rabbits. All animals received cyclosporine immunosuppression (10 mg/kg/day subcutaneously). The allografts were harvested at 3, 7, 10, 21, and 28 days after transplantation and assayed for beta-galactosidase activity., Results: Neointimal areas showed an initially slow increase for the first 10 days, followed by a rapid increase up to 21 days, and tended to plateau thereafter. Significant beta-galactosidase was apparent in aortic sections dissected from host rabbits for all time points, except at 28 days. At the 21-day time point, the aortic section from one rabbit was positive, whereas the other two remained negative. However, the one positive section showed intense beta-galactosidase activity, suggesting variability in the experimental model. At 28 days, all aortic sections were negative., Conclusions: Our findings confirm that genes delivered by this method are expressed for the duration of early rapid intimal proliferation in this heterotopic rabbit model of aortic allograft vasculopathy. These findings suggest that this animal model can be used to assess the therapeutic potential of gene transfer at the time of vascular transplantation and may provide a novel therapeutic approach to prevent or ameliorate the genesis of allograft vasculopathy.