Thoracic, Peripheral, and Cerebral Volume, Circulatory and Pressure Responses To PEEP During Simulated Hemorrhage in a Pig Model: a Case Study.

Positive end-expiratory pressure (PEEP) is a respiratory/ventilation procedure that is used to maintain or improve breathing in clinical and experimental cases that exhibit impaired lung function. Body fluid shift movement is not monitored during PEEP application in intensive care units (ICU), which would be interesting specifically in hypotensive patients. Brain injured and hypotensive patients are known to have compromised cerebral blood flow (CBF) autoregulation (AR) but currently, there is no non-invasive way to assess the risk of implementing a hypotensive resuscitation strategy and PEEP use in these patients. The advantage of electrical bioimpedance measurement is that it is noninvasive, continuous, and convenient. Since it has good time resolution, it is ideal for monitoring in intensive care units (ICU). The basis of its future use is to establish physiological correlates. In this study, we demonstrate the use of electrical bioimpedance measurement during bleeding and the use of PEEP in pig measurement. In an anesthetized pig, we performed multimodal recording on the torso and head involving electrical bioimpedance spectroscopy (EIS), fixed frequency impedance plethysmography (IPG), and bipolar (rheoencephalography - REG) measurements and processed data offline. Challenges (n=16) were PEEP, bleeding, change of SAP, and CO2 inhalation. The total measurement time was 4.12 hours. Systemic circulatory results: Bleeding caused a continuous decrease of SAP, cardiac output (CO), and increase of heart rate, temperature, shock index (SI), vegetative - Kerdo index (KI). Pulse pressure (PP) decreased only after second bleeding which coincided with loss of CBF AR. Pulmonary arterial pressure (PAP) increased during PEEP challenges as a function of time and bleeding. EIS/IPG results: Body fluid shift change was characterized by EIS-related variables. Electrical Impedance Spectroscopy was used to quantify the intravascular, interstitial, and intracellular volume changes during the application of PEEP and simulated hemorrhage. The intravascular fluid compartment was the primary source of blood during hemorrhage. PEEP produced a large fluid shift out of the intravascular compartment during the first bleeding period and continued to lose more blood following the second and third bleeding. Fixed frequency IPG was used to quantify the circulatory responses of the calf during PEEP and simulated hemorrhage. PEEP reduced the arterial blood flow into the calf and venous outflow from the calf. Head results: CBF AR was evaluated as a function of SAP change. Before bleeding, and after moderate bleeding, intracranial pressure (ICP), REG, and carotid flow pulse amplitudes (CFa) increased. This change reflected vasodilatation and active CBF AR. After additional hemorrhaging during PEEP, SAP, ICP, REG, CFa signal amplitudes decreased, indicating passive CBF AR. 1) The indicators of active AR status by modalities was the following: REG (n=9, 56 %), CFa (n=7, 44 %), and ICP (n=6, 38 %); 2) CBF reactivity was better for REG than ICP; 3) REG and ICP correlation coefficient were high (R2 = 0.81) during CBF AR active status; 4) PRx and REGx reflected active CBF AR status. CBF AR monitoring with REG offers safety for patients by preventing decreased CBF and secondary brain injury. We used different types of bioimpedance instrumentation to identify physiologic responses in the different parts of the body (that have not been discussed before) and how the peripheral responses ultimately lead to decreased cardiac output and changes in the head. These bioimpedance methods can improve ICU monitoring, increase the adequacy of therapy, and decrease mortality and morbidity.

Structure of a hyperthermostable dimeric archaeal Rubisco from Hyperthermus butylicus.

The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon Hyperthermus butylicus is presented at 1.8 Šresolution. Previous structures of archaeal Rubisco have been found to assemble into decamers, and this oligomerization was thought to be required for a highly thermally stable enzyme. In the current study, H. butylicus Rubisco is shown to exist as a dimer in solution, yet has a thermal denaturation midpoint of 114°C, suggesting that high thermal stability can be achieved without an increased oligomeric state. This increased thermal stability appears to be due to an increased number of electrostatic interactions within the monomeric subunit. As such, H. butylicus Rubisco presents a well characterized system in which to investigate the role of assembly and thermal stability in enzyme function.

Measurement of Cerebral Blood Flow Autoregulation with Rheoencephalography: A Comparative Pig Study.

Neuromonitoring is performed to prevent further (secondary) brain damage by detecting low brain blood flow following a head injury, stroke or neurosurgery. This comparative neuromonitoring study is part of an ongoing investigation of brain bioimpedance (rheoencephalography-REG) as a measuring modality for use in both civilian and military medical settings, such as patient transport, emergency care and neurosurgery intensive care. In a previous animal study, we validated that REG detects cerebral blood flow autoregulation (CBF AR), the body's physiological mechanism that protects the brain from adverse effects of low brain blood flow (hypoxia/ischemia). In the current descriptive pig study, the primary goal was to compare measurements of CBF AR made with REG to measurements made with other neuromonitoring modalities: laser Doppler flow (LDF); intracranial pressure (ICP); absolute CBF; carotid flow (CF); and systemic arterial pressure (SAP). Challenges administered to anesthetized pigs were severe induced hemorrhage (bleeding) and resuscitation; CO2 inhalation; and positive end expiratory pressure (PEEP). Data were stored on a computer and processed offline. After hemorrhage, the loss of CBF AR was detected by REG, ICP, and CF, all of which passively followed systemic arterial SAP after bleeding. Loss of CBF AR was the earliest indicator of low brain blood flow: loss of CBF AR occurred before a decrease in cardiac output, which is the cardiovascular response to hemorrhage. A secondary goal of this study was to validate the usefulness of new automated data processing software developed to detect the status of CBF AR. Both the new automated software and the traditional (observational) evaluation indicated the status of CBF AR. REG indicates the earliest breakdown of CBF AR; cessation of EEG for 2 seconds and respiration would be used as additional indicators of loss of CBF AR. The clinical significance of this animal study is that REG shows potential for use as a noninvasive, continuous and non-operator dependent neuromonitor of CBF AR in both civilian and military medical settings. Human validation studies of neuromonitoring with REG are currently in progress.

Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci.

Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10 These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations.

Characterization of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms reveals hexameric assemblies with increased thermal stability.

Most plants contain two isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a chloroplast protein that maintains the activity of Rubisco during photosynthesis. The longer (α-) Rca isoform has previously been shown to regulate the activity of Rubisco in response to both the ADP:ATP ratio and redox potential via thioredoxin-f. We have characterized the arrangement of the different spinach (Spinacia oleracea) isoforms in solution, and show how the presence of nucleotides changes the oligomeric state. Although the shorter (ß-) isoform from both tobacco (Nicotiana tabacum) and spinach tend to form a range of oligomers in solution, the size of which are relatively unaffected by the addition of nucleotide, the spinach α-isoform assembles as a hexamer in the presence of adenosine 5'-[γ-thio]triphosphate (ATPγS). These hexamers have significantly higher heat stability, and may play a role in optimizing photosynthesis at higher temperatures. Hexamers were also observed for mixtures of the two isoforms, suggesting that the α-isoform can act as a structural scaffold for hexamer formation by the ß-isoform. Additionally, it is shown that a variant of the tobacco ß-isoform acts in a similar fashion to the α-isoform of spinach, forming thermally stable hexamers in the presence of ATPγS. Both isoforms had similar rates of ATP hydrolysis, suggesting that a propensity for hexamer formation may not necessarily be correlated with activity. Modelling of the hexameric structures suggests that although the N-terminus of Rca forms a highly dynamic, extended structure, the C-terminus is located adjacent to the intersubunit interface.

Cessation of vital signs monitored during lethal hemorrhage: a Swine study.

INTRODUCTION: Two challenges of trauma triage are to identify wounded who are in danger of imminent death and to enable medics to determine if resuscitation is possible when making ?dead or alive? decisions on the battlefield. Hemorrhagic shock is the leading cause of death in combat injuries. The purpose of this study was to establish the sequence of vital sign cessation during lethal hemorrhage in swine. Our hypothesis was that brain electrical activity (electroencephalography [EEG]) and respiration are earlier indicators of imminent death than traditional modalities measured during triage, such as heart electrical activity (electrocardiography [ECG]) and blood pressure. METHODS: Lethal hemorrhage was induced in anesthetized Yorkshire pigs. Vital sign modalities measured were respiration, heart electrical activity (ECG), heart sound, blood pressure (systemic arterial pressure), and brain electrical activity (EEG). RESULTS: The sequence of vital sign cessation was (1) respiration, (2) brain electrical activity (EEG), (3) heart sound, (4) blood pressure, and (5) heart electrical activity (ECG). Cessation of respiration occurred at approximately the same time that brain electrical activity stopped (?flatlined?) for 2 seconds and then resumed briefly before cessation; cessation of heart electrical activity occurred almost 8 minutes later. CONCLUSIONS: A 2-second EEG flatline and final respiration are useful event markers to indicate an opportunity to prevent irreversible brain damage from lethal hemorrhage. Since the 2-second EEG flatline and final respiration occur about 8 minutes before cessation of heart electrical activity (ECG), EEG and final respiration are earlier indicators of imminent death. The use of deployable noninvasive brain monitors implementing these findings can be live-saving on the battlefield as well is in civilian environments.

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.

Cardiovascular responses to oxygen inhalation after hemorrhage in anesthetized rats: hyperoxic vasoconstriction.

Oxygen inhalation is recommended for the initial care of trauma victims. The improved survival seen in early hemorrhage is normally associated with an increase in blood pressure. Although clinical use of oxygen can occur late after hemorrhage, the effects of late administration have not been specifically examined. Anesthetized rats were studied using an isobaric hemorrhage model with target pressures of either 70 or 40 mmHg. At various times after hemorrhage, the feedback control of the blood pressure was stopped and the inspired gas was changed from room air to 100% oxygen. The results show that shortly after hemorrhage to 70 mmHg, oxygen inhalation results in an increase in mean arterial blood pressure of 60 +/- 3 mmHg, which is associated with a large increase in total peripheral resistance from 0.89 +/- 0.05 to 1.25 +/- 0.1 peripheral resistance units. The blood pressure response is essentially unchanged with time, and it is not altered by a 10-min exposure to N(G)-nitro-l-arginine methyl ester. At a target pressure of 40 mmHg, the initial blood pressure response to oxygen is the same, but it gradually decreases as the animal develops a lactic acidosis. We conclude that the therapeutic value of oxygen needs to be separately evaluated for late hemorrhage.

Impact of vasopressin on hemodynamic and metabolic function in the decompensatory phase of hemorrhagic shock.

OBJECTIVES: To explore how the potent vasoconstrictive features of vasopressin impact the rate of cardiovascular collapse and metabolic derangements associated with prolonged hemorrhagic shock. DESIGN: A prospective randomized trial. SETTING: University hospital-based animal laboratory. PARTICIPANTS: Sixteen swine. INTERVENTIONS: Swine were bled in an isobaric fashion to achieve a linear decrease in the mean arterial blood pressure to 40 mmHg. The mean arterial blood pressure was then maintained at 40 mmHg until the onset of cardiovascular decompensation, defined as the need to reinfuse shed blood to maintain the blood pressure at 40 mmHg. Once at the onset of cardiovascular decompensation, animals were randomly assigned to 2 resuscitation groups: the crystalloid group received lactated Ringer's solution and the vasopressin group received lactated Ringer's solution and arginine vasopressin. Resuscitation consisted of infusing lactated Ringer's solution with and without vasopressin (0.05 U/kg/min) to maintain a blood pressure of 70 mmHg for 60 minutes. MEASUREMENTS AND MAIN RESULTS: The rate of crystalloid infusion was compared between groups using an unpaired 2-tailed t test. Metabolic and hemodynamic parameters between groups over time were compared with a repeated measures analysis of variance. Vasopressin decreased the rate of crystalloid infusion during resuscitation by 50%. During resuscitation, the cardiac index in the crystalloid group was restored to near baseline levels and was decreased to near half of baseline levels in the vasopressin group. Animals in the vasopressin group developed a lactic acidemia, but animals in the crystalloid group revealed no change from baseline in the arterial pH and a slight decrease in the plasma lactate. CONCLUSIONS: Administration of vasopressin used as an adjunct to maintain blood pressure in the decompensatory phase of hemorrhagic shock slows cardiovascular collapse, but has an adverse effect on metabolic and hemodynamic function. Further investigation is warranted to clarify the role of vasopressin in the delayed management of severe hemorrhagic shock.

Cerebrovascular involvement in liposome-induced cardiopulmonary distress in pigs.

Intravenous administration of liposomes, including Doxil, can cause severe life-threatening hemodynamic changes in pigs. The reaction is due to complement activation, and it is characterized by massive pulmonary hypertension, systemic hypotension, and severe cardiac abnormalities including falling cardiac output, tachy-or bradycardia with arrhythmia. There were no data suggesting the involvement of cerebrovascular changes in this reaction; however, clinical observations allowed this hypothesis. Here we measured the accompanying changes during liposome infusion by monitoring pulsatile electrical impedance (rheoencephalogram- REG) on the skull (n=24 pigs, 57 trials, 19 types of liposomes). A transient but significant decrease of REG pulse amplitudes followed the injection of liposomes (78.43% in the total sample, and 91.66% in the Doxil subgroup; P=0.003, n=12), indicating the involvement of cerebrovascular reaction during liposome infusion.

Characterization of circulating monocytes expressing HLA-DR or CD71 and related soluble factors for 2 weeks after severe, non-thermal injury.

BACKGROUND: Severe injury is associated with changes in monocytes that may contribute to poor outcomes. Longitudinal characterization of monocyte response patterns after trauma may provide added insight into these immunological alterations. METHODS: Venous blood obtained seven times during post-injury days 1 through 13 from 61 patients with an injury severity score >20 was assessed by flow cytometry for monocytes (CD14+) expressing HLA-DR or CD71 (transferrin receptor) and for circulating levels of interleukin (IL) 1alpha, IL-1beta, IL-6, soluble CD14 (sCD14), tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), and endotoxin. Urine neopterin was measured by high-pressure liquid chromatography, expressed as a neopterin-creatinine ratio. RESULTS: Trauma patients had leucocytosis days 1 through 13, monocytosis days 5 through 13, reduced proportions of CD14+HLA-DR+ cells days 2 through 5, and elevated proportions of CD14+CD71+ cells days 1 through 13. Neopterin was elevated all days, peaking on day 10. sCD14 was elevated days 2 through 13, and there were sporadic elevations of IL-1alpha, IL-1beta, IL-6, TNF-alpha, PGE(2), TXB(2), and endotoxin. Sepsis syndrome patients (n = 6) had larger and more prolonged reductions in CD14+HLA-DR+ cells and higher neopterin values, in comparison with uneventful patient outcomes. CONCLUSIONS: Altered proportions of monocytes expressing HLA-DR and CD71 and elevated sCD14 and urine neopterin levels, for up to 2 weeks after severe injury, underscores an extended period of profound immunological effects. Additional studies to more fully assess temporal monocyte response patterns after severe injury, including activation, may be warranted.

Subcutaneous PO2 as an index of the physiological limits for hemodilution in the rat.

This study was designed to test the hypothesis that changes in subcutaneous PO2 (PscO2) during progressive hemodilution will reliably predict a "critical point" at which tissue O2 consumption (VO2) becomes dependent on O2 delivery (QO2). Twelve pentobarbital-anesthetized male Sprague-Dawley rats (315-375 g) underwent stepwise exchange of plasma for blood (1.5 ml of plasma for each 1 ml of blood lost). The initial exchange was equal to 25% of the estimated circulatory blood volume, and each subsequent exchange was equal to 10% of the estimated circulatory blood volume. After nine exchanges, the hematocrit (Hct) fell from 42 +/- 1 to 6 +/- 1%. Cardiac output and O2 extraction rose significantly. PscO2 became significantly reduced (P < 0.05) after exchange of 45% of the blood volume (Hct = 16 +/- 1%). VO2 became delivery dependent when QO2 fell below 21 ml x min(-1) x kg body wt(-1) (mean Hct = 13 +/- 1%). Eight control rats undergoing 1:1 blood-blood exchange showed no change in PscO2, pH, HCO3(-), or hemodynamics. Measurement of PscO2 may be a useful guide to monitor the adequacy of QO2 during hemodilution.

Measurement of brain electrical impedance: animal studies in rheoencephalography.

INTRODUCTION: In spite of the known importance of cerebral blood flow (CBF) monitoring for aviation, spaceflight, military and emergency medicine, and neurosurgical intra- and postoperative monitoring, there is no standard noninvasive technique for continuous CBF monitoring. One potential method for this purpose is the electrical impedance technique, called rheoencephalography (REG). The development of improved electronics and computation tools has done much to overcome the difficulties of REG measurement. REG technology now has possibilities for application to the fields mentioned above. HYPOTHESIS: Our hypothesis was that REG would reflect CBF changes. METHODS: Three experimental studies were undertaken to further define in vivo (rat, pig) CBF measurements by analysis of REG pulse waves. CO2 inhalation (4-20%), brain electrical stimulation, and aorta compression (5 min) were the applied CBF manipulations. In the case of aorta compression, global CBF was measured by REG, and local CBF by the laser Doppler method. Data were digitized and processed off-line. RESULTS: During CO2 inhalation and electrical stimulation of the brain, REG amplitude increased, indicating increased cerebral fluid volume. A linear relationship was established between CO2 concentration and REG peak amplitude (correlation coefficient: 0.88, p = 0.05), and the ascending portion of the curve (0.88, p = 0.05). During aorta compression, systemic arterial pressure increased (p = 0.008), and REG amplitude decreased (-23.75%, p = 0.01). CONCLUSION: These studies have confirmed the REG amplitude changes during known CBF manipulations. The difference between local and global CBF response demonstrated CBF autoregulation and heterogeneity. Together, these studies indicate the usefulness and potential benefit of computerized REG monitoring for the above-mentioned fields.

Employment retention of health care providers in frontier areas of Alaska.

OBJECTIVES: The objectives of this study were to: describe the length of employment of health care providers in rural Alaska; assess whether there are differences in length of employment among community health aides, medical doctors, and nurses; and determine whether provider length of employment is significantly increased following implementation of telemedicine. STUDY DESIGN: We conducted a prospective cohort study of length of employment among health professionals in rural Alaska, and identified the cohort based on current employment status of community health aides, medical doctors, and nurses. METHODS: Employment data were collected from four Alaska Native regional health corporations. Kaplan-Meier product-limit survival analysis was used to assess employment length. The Mantel-Haenszel log-rank test was used to test the difference between retention (survival) curves among doctors, nurses, and community health aides for all four regional health corporations combined. Data included provider hire date, termination date, and position title. Fifty seven percent of the data points were right-hand censored. RESULTS: The community health aides (median (Mdn) = 1186 days) were retained significantly longer than either the doctors (Mdn = 596 days), or the nurses (Mdn = 408 days), who were not significantly different from each other (log-rank chi2 (2, N = 996) = 68.30, p = 0.0001). CONCLUSIONS: Our findings document that community health aides in the region retain their jobs significantly longer than doctors and nurses. Findings highlight the problem of providing an adequate health work force in rural areas.

Clinical evaluation of the Life Support for Trauma and Transport (LSTAT) platform.

INTRODUCTION: The Life Support for Trauma and Transport (LSTAT trade mark ) is a self-contained, stretcher-based miniature intensive care unit designed by the United States Army to provide care for critically injured patients during transport and in remote settings where resources are limited. The LSTAT contains conventional medical equipment that has been integrated into one platform and reduced in size to fit within the dimensional envelope of a North Atlantic Treaty Organization (NATO) stretcher. This study evaluated the clinical utility of the LSTAT in simulated and real clinical environments. Our hypothesis was that the LSTAT would be equivalent to conventional equipment in detecting and treating life-threatening problems. METHODS: Thirty-one anesthesiologists and recovery room nurses compared the LSTAT with conventional monitors while managing four simulated critical events. The time required to reach a diagnosis and treatment was recorded for each simulation. Subsequently, 10 consenting adult patients were placed on the LSTAT after surgery for postoperative care in the recovery room. Questionnaires about aspects of LSTAT functionality were completed by nine nurses who cared for the patients placed on the LSTAT. RESULTS: In all of the simulations, there was no clinically significant difference in the time to diagnosis or treatment between the LSTAT and conventional equipment. All clinicians reported that they were able to manage the simulated patients properly with the LSTAT. Nursing staff reported that the LSTAT provided adequate equipment to care for the patients monitored during recovery from surgery and were able to detect critical changes in vital signs in a timely manner. DISCUSSION: Preliminary evaluation of the LSTAT in simulated and postoperative environments demonstrated that the LSTAT provided appropriate equipment to detect and manage critical events in patient care. Further work in assessing LSTAT functionality in a higher-acuity environment is warranted.

Effect of Misoprostol on Histamine Secretion from Isolated Rat and Human Mast Cells and Basophils.

Misoprostol was found to be an extremely potent inhibitor of immunologically induced histamine release from mast cells isolated from both the human colonic mucosa and submucosa/muscle. The dose-inhibition curves were bell shaped, with IC(50) values in the range 0.1--1 nM. The compound was relatively selective for these cell types. It was less active against rat peritoneal mast cells, which again exhibited a bell-shaped dose-inhibition curve, but with a maximal inhibition of only about 30%, and human basophil leucocytes, where the inhibition was progressive with an IC(50) of similar1 &mgr;M. Very high concentrations of misoprostol (greater-than-or-equal5 &mgr;M) induced histamine release from all of the cell types examined. Insofar as the colonic mast cells may represent the two human mast cell phenotypes so far identified (MC(T) and MC(TC)), these data suggest that the drug may have therapeutic applications in human allergic and inflammatory diseases in which these cells are involved.