Noninvasive neuromonitoring with rheoencephalography: a case report.

Cerebral blood flow (CBF) autoregulation (AR) can be monitored using invasive modalities, such as intracranial pressure (ICP) and arterial blood pressure (ABP) to calculate the CBF AR index (PRx). Monitoring PRx can reduce the extent of secondary brain damage in patients. Rheoencephalography (REG) is an FDA-approved non-invasive method to measure CBF. REGx, a CBF AR index, is calculated from REG and arm bioimpedance pulse waves. Our goal was to test REG for neuromonitoring. 28 measurement sessions were performed on 13 neurocritical care patients. REG/arm bioimpedance waveforms were recorded on a laptop using a bioimpedance amplifier and custom-built software. The same program was used for offline data processing. Case #1: The patient's mean REGx increased from - 0.08 on the first day to 0.44 on the second day, indicating worsening intracranial compliance (ICC) (P < 0.0001, CI 0.46-0.58). Glasgow Coma Scale (GCS) was 5 on both days. Case #2: REGx decreased from 0.32 on the first recording to 0.07 on the last (P = 0.0003, CI - 0.38 to - 0.12). GCS was 7 and 14, respectively. Case #3: Within a 36-minute recording, REGx decreased from 0.56 to - 0.37 (P < 0.0001, 95%, CI - 1.10 to - 0.76). Central venous pressure changed from 14 to 9 mmHg. REG pulse wave morphology changed from poor ICC to good ICC morphology. Bioimpedance recording made it possible to quantify the active/passive status of CBF AR, indicate the worsening of ICC, and present it in real time. REGx can be a suitable, non-invasive alternative to PRx for use in head-injured patients.

The effect of the perfluorocarbon emulsion Oxycyte™ in an ovine model of severe decompression illness.

Background: The treatment of decompression sickness (DCS) with hyperbaric oxygen (HBO2) serves to decrease intravascular bubble size, increase oxygen (O2) delivery to tissue and enhance the elimination of inert gas. Emulsified perfluorocarbons (PFC) combined with breathing O2 have been shown to have similar effects animal models. We studied an ovine model of severe DCS treated with the intravenous PFC Oxycyte™ while breathing O2 compared to saline control also breathing O2. Methods: Juvenile male sheep (N=67; weight 24.4±2.10kg) were compressed to 257 feet of sea water (fsw) in our multiple large-animal chamber where they remained under pressure for 31 minutes. Animals then were decompressed to surface pressure and randomized to receive either Oxycyte at 5mL/kg intravenously (IV) or 5mL/kg saline IV (both receiving 100% O2) 10 minutes after reaching surface pressure. Mortality was recorded at two hours, four hours, and 24 hours after receiving the study drug. Surviving animals underwent perfusion fixation and harvesting of the spinal cord at 24 hours. Spinal cord sections were assessed for volume of lesion area and compared. Results: There was no significant difference in survival at two hours (p=0.2737), four hours (p=0.2101), or 24 hours (p=0.3171). Paralysis at 24 hours was not significantly different. However, spinal cord lesion area was significantly smaller in the Oxycyte group as compared to the saline group, with median spinal cord lesion areas 0.65% vs. 0.94% (p=0.0107). Conclusion: In this ovine model of severe DCS the intravenous PFC Oxycyte did not reduce mortality but did ameliorate spinal cord injury when used after the onset of DCS.

Rheoencephalography: A non-invasive method for neuromonitoring.

In neurocritical care, the gold standard method is intracranial pressure (ICP) monitoring for the patient's lifesaving. Since it is an invasive method, it is desirable to use an alternative, noninvasive technique. The computerized real-time invasive cerebral blood flow (CBF) autoregulation (AR) monitoring calculates the status of CBF AR, called the pressure reactivity index (PRx). Studies documented that the electrical impedance of the head (Rheoencephalography - REG) can detect the status of CBF AR (REGx) and ICP noninvasively. We aimed to test REG to reflect ICP and CBF AR. For nineteen healthy subjects we recorded bipolar bifrontal and bitemporal REG derivations and arm bioimpedance pulses with a 200 Hz sampling rate. The challenges were a 30-second breath-holding and head-down-tilt (HDT - Trendelenburg) position. Data were stored and processed offline. REG pulse wave morphology and REGx were calculated. The most relevant finding was the significant morphological change of the REG pulse waveform (2nd peak increase) during the HDT position. Breath-holding caused REG amplitude increase, but it was not significant. REGx in male and female group averages have similar trends during HDT by indicating the active status of CBF AR. The morphological change of REG pulse wave during HDT position was identical to ICP waveform change during increased ICP, reflecting decreased intracranial compliance. A correlation study between ICP and REG was initiated in neurocritical care patients. The noninvasive REG monitoring would also be useful in space research as well as in military medicine during the transport of wounded service members as well as for fighter pilots to indicate the loss of CBF and consciousness.

The intravenous perfluorocarbon emulsion Oxycyte does not increase hyperbaric oxygen-related seizures in a non-sedated swine model.

PURPOSE: Standard treatment for decompression sickness (DCS) is recompression therapy with hyperbaric oxygen (HBO). Non-recompressive therapies are needed to address mass casualty scenarios such as a disabled submarine rescue or DCS therapy in remote environments. Intravenously delivered perfluorocarbon (PFC) emulsions improve blood oxygen content and decrease mortality in several animal models of DCS. However, the enhanced oxygen delivery of PFC emulsions may increase CNS oxygen toxicity (seizures) risk when used in conjunction with HBO. We studied seizure latency and duration in swine randomized to receive PFC or normal saline with 6 ATA of oxygen. METHODS: Yorkshire swine (n = 31) were fitted with EEG electrodes and randomized to receive 5 ml/kg of the PFC Oxycyte (Oxygen Biotherapeutics Inc., Morrisville, NC) or saline intravenously 1 h before HBO. Unsedated animals were fitted with a snout mask for inhaled gas delivery, positioned inside the hyperbaric chamber, and compressed to 165 ft of sea water (6 ATA). After 2.5 min at 6 ATA, breathing gas was switched to 100 % O2 until signs of seizure were observed and EEG activity was evident. At seizure onset gas was switched back to air for 3 min, then the chamber was decompressed. After 24 h, the dive profile/oxygen exposure was repeated to ensure no secondary effects of PFC drug redistribution or emulsion metabolism. RESULTS/CONCLUSION: Intravenous PFC emulsion did not decrease seizure latency or increase duration on initial HBO exposure or after 24 h. This finding demonstrates the safety of PFC use in conjunction with recompression therapy to treat DCS.

Multimodal noninvasive monitoring of soft tissue wound healing.

Here we report results of non-invasive measurements of indirect markers of soft tissue healing of traumatic wounds in an observational swine study and describe the quantification of analog physiological signals. The primary purpose of the study was to measure bone healing of fractures with four different wound treatments. A second purpose was to quantify soft tissue wound healing by measuring the following indirect markers: (1) tissue oxygenation, (2) fluid content, and (3) blood flow, which were all measured by non-invasive modalities, measured with available devices. Tissue oxygenation was measured by near infrared spectroscopy; fluid content was measured by bipolar bio-impedance; and blood flow was measured by Doppler ultrasound. Immediately after comminuted femur fractures were produced in the right hind legs of thirty anesthetized female Yorkshire swine, one of four wound treatments was instilled into each wound. The four wound treatments were as follows: salmon fibrinogen/thrombin-n = 8; commercial bone filler matrix-n = 7; bovine collagen-n = 8; porcine fibrinogen/thrombin-n = 7. Fractures were stabilized with an external fixation device. Immediately following wound treatments, measurements were made of tissue oxygenation, fluid content and blood flow; these measurements were repeated weekly for 3 weeks after surgery. Analog signals of each modality were recorded on both the wounded (right) hind leg and the healthy (left) hind leg, for comparison purposes. Data were processed off-line. The mean values of 10-s periods were calculated for right-left leg comparison. ANOVA was applied for statistical analysis. Results of the bone healing studies are published separately (Rothwell et al. in J Spec Oper Med 13:7-18, 2013). For soft tissue wounds, healing did not differ significantly among the four wound treatments; however, regional oxygenation of wounds treated with salmon fibrinogen/thrombin showed slightly different time trends. Further studies are needed to establish standards for healthy wound healing and for detection of pathological alterations such as infection. Non-invasive measurement and quantification of indirect markers of soft tissue wound healing support the goals and principles of evidence-based medicine and show potential as easy to administer tools for clinicians and battlefield medical personnel to apply when procedures such as the PET scan are not available or affordable. The method we developed for storing analog physiological signals could be used for maintaining electronic health records, by incorporating vital signs such as ECG and EEG, etc.

Characteristics of Rheoencephalography and some Associated Factors on Menopausal Women.

The significant drop in estrogen levels during menopause increases the cardiovascular risks, one of which is cerebrovascular atherosclerosis. Research on rheoencephalography (REG) parameters for the early diagnosis of cerebrovascular atherosclerotic lesions is of great interest to scientists because of its ease of implementation, low cost, and non-invasiveness. The objectives of study are to evaluate the vascular tone, cerebral circulation flow in each hemisphere of the brain of menopausal women, and some associated factors through waveform characteristics and parameters in REG. A controlled cross-sectional descriptive study was conducted on a group of patients including 80 menopausal women and a control group of 46 menstruating women. All patients were measured REG in the frontal-occipital leads by VasoScreen 5000 impedance REG meter. In menopausal women, the percentage of sharp waves, the percentage of clear side waves, and the average REG were all lower than in the control group (p<0.01). The mean conduction time and mean slope ratio was lower than the control group (p<0.001). The mean peak time was higher than the control group (p<0.01). The mean elasticity index (alpha/T) was higher than the control group (p<0.001). Menopausal women have increased vascular tone, the highest in the group of women 50-60 years old, menopause <5 years, having a habit of eating red meat; and decreased blood flow intensity, the highest in the group of women <50 years old. However, the difference was statistically significant only in the left hemisphere (p<0.05). Vascular hypertonia in menopausal women with central obesity was higher than in the non-obese group in both hemispheres (p<0.05). In conclusion, menopausal women had atherosclerosis in both hemispheres of the brain, which was clearly shown in the rate of increased vascular tone. Central obesity may increase the risk of vascular hypertonia 3.75 times in the right and 5.44 times in the left hemisphere.

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.

Segmental Intracellular, Interstitial, and Intravascular Volume Changes during Simulated Hemorrhage and Resuscitation: A Case Study.

This paper describes a new combined impedance plethysmographic (IPG) and electrical bioimpedance spectroscopic (BIS) instrument and software that will allow noninvasive real-time measurement of segmental blood flow, intracellular, interstitial, and intravascular volume changes during various fluid management procedures. The impedance device can be operated either as a fixed frequency IPG for the quantification of segmental blood flow and hemodynamics or as a multi-frequency BIS for the recording of intracellular and extracellular resistances at 40 discrete input frequencies. The extracellular volume is then deconvoluted to obtain its intravascular and interstitial component volumes as functions of elapsed time. The purpose of this paper is to describe this instrumentation and to demonstrate the information that can be obtained by using it to monitor segmental compartment volume responses of a pig model during simulated hemorrhage and resuscitation. Such information may prove valuable in the diagnosis and management of rapid changes in the body fluid balance and various clinical treatments.

Risk of mental disorders, their changes and somatic consideration in rural Hungary.

OBJECTIVE: Although the primary purpose of the study reported here was to identify stroke risk factors among the residents of a village in eastern Hungary, the study also included a multi-faceted survey conducted in 1992-94 to ascertain the somatic, mental and socio-economic conditions of the residents. Here we report data from the survey on prevalence of mental disorders (a cross-sectional descriptive study). METHOD: The screenings included the following tests administered to 535 subjects: anxiety, depression, dementia, neurosis were measured; recent medical records were compared to survey data for 330 of the same subjects. FINDINGS: The summary of prevalence of mental disorders measured in this study was as follows: anxiety 34.7% (severe), dementia 44.68% (mild), depression 66% (mild), 15.94% (medium), 7.88% (severe), neurosis 66.73% (mild, medium, and severe). Medical records maintained by village physicians since 1960 differed from the results obtained in the present study. A treatment gap was observed between mental health treatment for neurosis, as indicated by medical records, and the diagnostic prevalence of neurosis as measured by the survey instruments: there were three times as many people diagnosed as neurotic in the survey as had been noted in village medical records. Additionally, the unique position of cerebrovascular alteration was established between the mental and somatic factors. CONCLUSION: The study demonstrates the successful simultaneous collection of a wide spectrum of data on somatic conditions, mental disorders, and socio-economic status of the subjects. The study showed that 1. patient-centered medical care can simultaneously address both somatic and mental factors; 2. it is possible to decrease the treatment gap in mental health; 3. there is value in systematic collection of data in order to optimize the planning of prevention, health care costs and decision making.

Prevalence of stroke/cardiovascular risk factors in rural Hungary--a cross-sectional descriptive study.

UNLABELLED: A multi-faceted survey was conducted in 1992-94 to ascertain the somatic, mental and socio-economic conditions of the residents of a village in eastern Hungary. Here we report data on prevalence of somatic disorders from the survey. OBJECTIVES: a) To collect and compare prevalence of known cardiovascular disease, including stroke risk factors, in a specific population (a Hungarian village); b) to test a computer-based mass screening device ("Cerberus") designed to identify individuals in the test population at high risk for stroke; c) to compare results obtained with Cerberus with known stroke risk/cardiovascular disease factors and traditional medical records. METHODS: A cross-sectional survey (546 subjects) was conducted in Csengersima in the early 1990s, using the Cerberus screening system, which includes: 1. a questionnaire addressing the risk factors for stroke/cardiovascular disease; 2. amplifiers to record the pulse waves of cerebral (rheoencephalography) and peripheral arteries, electrocardiogram and electroencephalogram. Additionally, subjects were measured for carotid stenosis by Doppler ultrasound and 12-lead electrocardiogram; they were also screened for blood cholesterol, glucose, and triglyceride levels. FINDINGS: Prevalence of the following stroke risk factors was identified: overweight, 63.25%; sclerotic brain arteries by rheoencephalogram, 54.29%; heart disease, 37.92%; pathologic carotid flow, 34.24%; smoking, 30.55%; high blood cholesterol, 28.70%; hypertension, 27.83%; high triglyceride, 24.35%; abnormality of electrocardiogram, 20%; high glucose, 15.95%; symptoms of transient ischemic attack, 16.07%; alcohol abuse, 6.74%; and diabetes, 4.53%. CONCLUSION: The study demonstrates a possible model for primary cardiovascular disease/stroke prevention. The simple, noninvasive test uses the bioimpedance method of measurement. This method offers a standardizable, cost-effective, practical technique for mass screenings by identifying the population at high risk for cardiovascular disturbances, especially cerebrovascular disease. In this model, the rheoencephalogram can detect cerebrovascular arteriosclerosis in the susceptibility/presymptomatic phase, earlier than the Doppler ultrasound technique. The method also provides a model for storing analog physiological signals in a computer-based medical record and the first steps of turning it into an expert system also tested.

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.

Recombinant collagen and gelatin for drug delivery.

The tools of recombinant protein expression are now being used to provide recombinant sources of both collagen and gelatin. The primary focus of this review is to discuss alternatives to bovine collagen for biomedical applications. Several recombinant systems have been developed for production of human sequence collagens. Mammalian and insect cells were initially used, but were thought to be too costly for commercial production. Yeast have been engineered to express high levels of type I homotrimer and heterotrimer and type II and type III collagen. Co-expression of collagen genes and cDNAs encoding the subunits of prolyl hydroxylase has lead to the synthesis of completely hydroxylated, thermostable collagens. Human types I and III collagen homotrimers have been expressed in transgenic tobacco plants, while transgenic mice have been engineered to produce full-length type I procollagen homotrimer as well as a alpha2 (I) homotrimeric mini-collagen. Most recently, a transgenic silkworm system was used to produce a fusion protein containing a collagenous sequence. Each of these transgenic systems holds great promise for the cost-effective large-scale production of recombinant human collagens. As seen in other recombinant expression systems, transgenic silkworms, tobacco, and mice lack sufficient endogenous prolyl hydroxylase activity to produce fully hydroxylated collagen. In mice and tobacco, this was overcome by over-expression of prolyl hydroxylase, analogous to what has been done in yeast and insect cell culture. In addition to recombinant alternatives to bovine collagen, other sources such as fish and sponge collagen are discussed briefly. Recombinant gelatin has been expressed in Pichia pastoris and Hansenula polymorpha in both non-hydroxylated and hydroxylated forms. Pichia was shown to be a highly productive system for gelatin production. The recombinant gelatins produced in yeast are of defined molecular weight and physio-chemical properties and represent a new biomaterial not previously available from animal sources. Genetic engineering has made great progress in the areas of recombinant collagen and gelatin expression, and there are now several alternatives to bovine material that offer an enhanced safety profile, greater reproducibility and quality, and the ability of these materials to be tailored to enhance product performance.

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.

Vigabatrin prevents seizure in swine subjected to hyperbaric hyperoxia.

Oxygen is the most widely used therapeutic strategy to prevent and treat decompression sickness (DCS). Oxygen prebreathe (OPB) eliminated DCS in 20-kg swine after rapid decompression from saturation at 60 feet of seawater (fsw). However, hyperbaric oxygen (HBO) has risks. As oxygen partial pressure increases, so do its toxic effects. Central nervous system (CNS) oxygen toxicity is the most severe side effect, manifesting as seizure. An adjunctive therapeutic is needed to extend OPB strategies to deeper depths and prevent/delay seizure onset. The Food and Drug Administration-approved anti-epileptic vigabatrin has prevented HBO-induced seizures in rats up to 132 fsw. This study aimed to confirm the rat findings in a higher animal model and determine whether acute high-dose vigabatrin evokes retinotoxicity symptoms seen with chronic use clinically in humans. Vigabatrin dose escalation studies were conducted 20-kg swine exposed to HBO at 132 or 165 fsw. The saline group had seizure latencies of 7 and 11 min at 165 and 132 fsw, respectively. Vigabatrin at 180 mg/kg significantly increased latency (13 and 27 min at 165 and 132 fsw, respectively); 250 mg/kg abolished seizure activity at all depths. Functional electroretinogram and histology of the retinas showed no signs of retinal toxicity in any of the vigabatrin=treated animals. In the 250 mg/kg group there was no evidence of CNS oxygen toxicity; however, pulmonary oxygen toxicity limited HBO exposure. Together, the findings from this study show that vigabatrin therapy is efficacious at preventing CNS oxygen toxicity in swine, and a single dose is not acutely associated with retinotoxicity.

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.

Comparison of fibrinogen- and collagen-based treatments for penetrating wounds with comminuted femur fractures in a Swine model.

INTRODUCTION: Military servicemembers in combat operations often sustain injuries to the extremities from highspeed projectiles, resulting in bleeding and comminuted open fractures. Severe injury with bone fragmentation can result in limb amputation. Surgical treatment options include materials that promote osteogenesis and bone proliferation, such as growth hormones, stem cells, or mineralized matrix adjuncts. However, none of these are amenable to use by the first responder, nor do they address the question of hemorrhage control, which is a common problem in traumatic injuries. HYPOTHESIS: Our hypothesis was that treatment with a fibrinogen-based protein mixture at the time of the bone injury will provide both hemostasis and a supportive environment for preservation of injured bone. METHODS: A comminuted femur fracture was produced in 28 female Yorkshire swine, and one of four treatments was instilled into the wound immediately after injury. Each animal was evaluated for the following parameters: inflammation, new bone growth, osteoclast proliferation, callus formation, and femur wound cavity fill, using post-mortem computed tomography and analysis of histological sections. RESULTS: Overall, salmon fibrinogen?thrombin and porcine fibrinogen?thrombin showed a trend for improved healing based on bone filling and calcification. However, statistically significant differences could not be established between treatment groups. CONCLUSIONS: These findings indicate that a fibrinogen?thrombin matrix may be a useful as an immediate response product to enhance fracture healing. Salmon fibrinogen?thrombin has the advantages of cost and a pathogen profile compared to mammalian fibrinogens.

Assessment of postoperative analgesia after application of ultrasound-guided regional anesthesia for surgery in a swine femoral fracture model.

Management of pain in research swine used for studies involving painful procedures is a considerable challenge. Here we assessed whether a regional anesthesia method is effective for pain control of hindlimb injuries in pigs used for research in bone fracture healing. For this randomized controlled study, we administered regional anesthesia before an experimental femoral injury was produced. Using ultrasound guidance, we placed sterile infusion catheters near the sciatic and femoral nerves and administered local anesthetic (bupivacaine) for the first 24 h after surgery. We evaluated various behavioral and physiologic parameters to test the hypothesis that this regional anesthesia would provide superior analgesia compared with systemic analgesia alone. We also collected blood samples to evaluate serum levels of cortisol and fentanyl postoperatively. At the end of the study period, we collected sciatic and femoral nerves and surrounding soft tissues for histopathologic evaluation. Treatment pigs had lower subjective pain scores than did control animals. Control pigs had a longer time to first feed consumption and required additional analgesia earlier in the postoperative period than did treatment pigs. Ultrasound-guided regional anesthesia is a viable and effective adjunct to systemic analgesics for providing pain control in swine with experimental femoral fractures.

A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor.

A fusion protein based expression system was developed in the Gram-positive bacterium Bacillus subtilis to produce the soybean Bowman-Birk protease inhibitor (sBBI). The N-terminus of the mature sBBI was fused to the C-terminus of the 1st cellulose binding domain linker (CBD linker) of the BCE103 cellulase (from an alkalophilic Bacillus sp.). The strong aprE promoter was used to drive the transcription of the fusion gene and the AprE signal sequence was fused to the mature BCE103 cellulase for efficient secretion of the fusion protein into the culture medium. It was necessary to use a B. subtilis strain deficient in nine protease genes in order to reduce the proteolytic degradation of the fusion protein during growth. The fusion protein was produced in shake flasks at concentrations >1g/L. After growth, the sBBI was activated by treatment with 2-mercaptoethanol to allow the disulfide bonds to form correctly. An economical and scalable purification process was developed to purify sBBI based on acid precipitation of the fusion protein followed by acid/heat cleavage of the fusion protein at labile Asp-Pro bonds in the CBD linker. If necessary, non-native amino acids at the N- and C-termini were trimmed off using glutamyl endopeptidase I. After purification, an average of 72 mg of active sBBI were obtained from 1L of culture broth representing an overall yield of 21% based on the amount of sBBI activated before purification.

Complement activation-related cardiac anaphylaxis in pigs: role of C5a anaphylatoxin and adenosine in liposome-induced abnormalities in ECG and heart function.

Cardiac anaphylaxis is a severe, life-threatening manifestation of acute hypersensitivity reactions to allergens and drugs. Earlier studies highlighted an amplifying effect of locally applied C5a on the process; however, the role of systemic complement (C) activation with C5a liberation in blood has not been explored to date. In the present study, we used the porcine liposome-induced cardiopulmonary distress model for 1) characterizing and quantifying peripheral C activation-related cardiac dysfunction; 2) exploring the role of C5a in cardiac abnormalities and therapeutic potential of C blockage by soluble C receptor type 1 (sCR1) and an anti-C5a antibody (GS1); and 3) elucidating the role of adenosine and adenosine receptors in paradoxical bradycardia, one of the symptoms observed in this model. Pigs were injected intravenously with different liposomes [Doxil and multilamellar vesicles (MLV)], zymosan, recombinant human (rhu) C5a, and adenosine, and the ensuing hemodynamic and cardiac changes (hypotension, tachy- or bradycardia, arrhythmias, ST-T changes, ventricular fibrillation, and arrest) were quantified by ranking on an arbitrary scale [cardiac abnormality score (CAS)]. There was significant correlation between CAS and C5a production by liposomes in vitro, and the liposome-induced cardiac abnormalities were partially or fully reproduced with zymosan, rhuC5a, adenosine, and the selective adenosine A1 receptor agonist cyclopentyl-adenosine. The use of C nonactivator liposomes or pretreatment of pigs with sCR1 or GS1 attenuated the abnormalities. The selective A1 blocker cyclopentyl-xanthine inhibited bradycardia without influencing hypotension, whereas the A(2) blocker 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-24135) had no such effect. These data suggest that 1) systemic C activation can underlie cardiac anaphylaxis, 2) C5a plays a causal role in the reaction, 3) adenosine action via A1 receptors may explain paradoxical bradycardia, and 4) inhibition of C5a formation or action or of A1-receptor function may alleviate the acute cardiotoxicity of liposomal drugs and other intravenous agents that activate C.