VCU

Research Frontiers at the Virginia Commonwealth University Department of Anesthesiology Laboratories

Perfluorocarbon Emulsions as a Treatment/Prevention for Arterial/Venous Gas Embolism, Decompression Illness and State of the Art Bubble Detection Technologies

Perfluorocarbon emulsions (PFC) are pharmaceutical intravenous lipid micelle emulsions containing one or more straight chain or cyclic carbon chains with all available side chains halogenated. These synthetic oils in nano-particle sizes have enhanced non-polar gas (O2, CO2, N2, NO, CO, etc.) solubility properties allowing for increased O2 delivery from erythrocytes to tissues in the microcirculation. Older work had explored these agents as “blood substitutes” with a belief that PFCs could function well as a “third compartment” in the blood for gas transport. Our laboratory has taken a fundamentally different view of these emulsions, embracing them as potential pharmaceutical agents capable of enhanced gas transport and diffusion, not as a replacement for blood transfusion. PFCs carry N2 , the source of emboli associated with decompression illness (DCI), and they carry O2. Our laboratories had 12 years of funding by the Office of Naval Research (ONR) and in collaboration with Navy Undersea/Dive Medicine in Bethesda, Maryland. During those years (2002-2013) a series of sheep, swine and rabbit studies were performed with experimental venous gas embolism (VGE) and arterial gas embolism (AGE). In total the work has demonstrated that: A) PFC pre-treatment will reduce the severity and will speed the recovery from VGE and AGE. B) In severe DCI, PFC treatment at surface (within 10 minutes) dramatically reduces (>60%) lethality from cardiopulmonary DCI (the “chokes”). C) In spinal cord or cerebral AGE, PFC treatment preserves brain and spinal cord tissue otherwise at high risk for neurologic injury. D) In our microcirculation laboratories a major discovery was made when we observed that, when red blood cell delivery of O2 to downstream tissue was blocked by AGE or mechanical obstruction, PFC present in the trickle flow of plasma was sufficient to maintain tissue oxygenation in the absence of red blood cells. We believe this finding alone can be a potential revolution for medicine. The implications of having an intravenous fluid capable of increasing tissue gas delivery in the face of marginal to no blood flow begs a great deal of future research. The discovery that PFC treatment can offer a non-recompressive (normobaric) alternative for either surgical gas embolism or DCI has great clinical promise and is awaiting FDA approval for human trials. In parallel studies of DCI in large animal models, our group is characterizing the efficacy of PFC treatment by quantifying venous and arterial bubble load and its correlation with outcome. PFC will decrease bubble load by enhancing N2 solubility and off-gassing in the lungs. DCI is mostly associated with VGE, but more serious AGE can occur. Utilizing ultrasound probes, bubbles can be simultaneously quantified in the jugular vein and carotid artery to assess at what venous bubble load arterial bubbles begin to manifest. At the same time, a fundus camera is used to ascertain bubble load in the retinal circulation. The retinal circulation is a noninvasive, real-time window into the microcirculation of the brain. Data suggest that retinal bubble load correlates very well with outcome following rapid decompression. This tool is being developed as an important aid in the triage of patients in a decompression emergency scenario.

Grants:
1. ONR, U S DoD ($950,754.00 over 3 years) - Coordinated studies in the mechanisms of organ protection from decompression sickness with a perfluorocarbon emulsion. March 2002-2005.

2. Phase I SBIR Luna Innovations/ONR ($75,000.00) - Development of a high fidelity tissue bubble detection system for DCS. Luna Innovations Inc., Hampton Roads, VA.

3. ONR, U S DoD-DURIP Award ($120,000.00) - Retinal Fluorescein Angiography Studies of Decompression Sickness: May 1, 2004.

4. ONR, U S DoD ($1,311,505.00) - Follow-on Studies in Perfluorocarbon Emulsions, Prevention and Treatment of Decompression Sickness. March 31, 2005 to March 30, 2008.

5. ONR, U S DoD ($224,531.00) - Post Doctoral Award to Study Methods of Detection and Assessment of Decompression Sickness, Prevention and Treatment with Perfluorocarbon Emulsions: June 1, 2006 to May 31, 2008.

6. Phase II SBIR ONR: Further Development of an ultrasound/sonar tissue bubble detection system for DCS ($1,000,000.00) - Luna Innovations, Hampton Roads, Virginia. August 2007. Subcontract Award to VCURES.

7. Sponsor: ONR, U S DoD: Grant # N000140810462
Title: Study of the Microcirculation in Decompression Sickness (DCS), Arterial Gas Embolism (AGE) and Traumatic Brain Injury (TBI) with and without Treatment using a Perfluorocarbon Based Oxygen Therapeutic.
Dates: 02/08-03/2011.
Costs: Total Award: $337,825.00.
Description: This is the award for 3 years of training for a postdoctoral fellow.

8. Sponsor: ONR, U S DoD: Grant # N000140810459
Title: Mechanistic and Monitoring Studies in Experimental Decompression Sickness (DCS) Utilizing Novel Treatments with Perfluorocarbon (PFC) Emulsions.
Dates: 02/08-03/2011.
Costs: Total Award: $1,218,728.00. Indirect Costs: $381,058.00.
Description: This is the award for continuous study of experimental DCS treated with PFC.

9. Sponsor: ONR, U S DoD: Grant # N000140810474
Title: Arterial Gas Embolism: Diagnosis and Novel Therapeutic of Perfluorocarbon Emulsions.
Dates: 02/08-03/2011.
Costs: Total Award: $1,671,586.00. Indirect Costs: $541,495.00.
Description: This is the award for the study of the effect of PFC on arterial gas embolism.

10. Sponsor: ONR: N00014-07-M-0430 Subcontract number: 1766-NVY-1T/VCU
Title: Ultrasonic Measure of the Formation of Bubbles in Tissue for Decompression Sickness.
Dates: 08/07 - 02/2008.
Costs: Total Award: $21,000.00 Indirect Costs: $6,906.00.
Description: This is the award for the study of the effect of PFC on arterial gas embolism.

References:

  1. Torres Filho IP, Torres LN, Spiess BD. In vivo microvascular mosaics show air embolism reduction after perfluorocarbon emulsion treatment. Microvasc Res. 2012; 84: 390-4. PMID: 23010091.
  2. Smith CR, Parsons JT, Zhu J, Spiess BD. The effect of intravenous perfluorocarbon emulsions on whole-body oxygenation after severe decompression sickness. Diving and Hyperbaric Medicine 2012; 42: 10-17.
  3. Torres LN, Spiess, BD, Torres- Filho IP. Tissue oxygenation and microvascular hemodynamics in experimental arterial gas embolism. Undersea Hyperb Med 2011; 38: 537-48.
  4. Smith CR, Spiess BD. The two faces of Eve; gaseous anaesthesia and inert gas narcosis. Div Hyperb Med 2010; 40: 68-77.
  5. Spiess BD.Basicmechanisms of gas transport and past research using perfluorocarbons. Diving Hyperbaric Med2010; 40: 23-28.
  6. Spiess BD. The potential role of perfluorocarbon emulsions in decompression illness. Diving Hyperbaric Med 2010; 40:28-33.
  7. Parsons JT, Smith CR, Zhu J, Spiess BD. Retinal angiography: Noninvasive, real-time bubble assessment from the ocular fundus. Undersea Hyperbar Med 2009; 36: 169-81.
  8. Spiess BD. Perfluorocarbon emulsions as a promising technology: a review of tissue and vascular gas dynamics. J Appl Physiol 2009; 106:1444-52. (Epub ahead of print) April 2009.
  9. Torres Filho I P, Torres LN, Spiess BD, Microvascular hemodynamics and oxygenation during experimental gas embolism. Proceedings of the 25th Conference of the European Society of Microcirculation, Medimond 2008; 1-5, K826c0264.
  10. Spiess BD, Zhu J, Pierce B, Weis R, Berger BE, Reses J, Smith CR, Ewbank B, Ward KR. Effects of perfluorocarbon infusion in an anesthetized swine decompression model. J Surg Res 2008; Mar 26 (E Pub ahead of print) PMID: 18541265.
  11. Zhu J, Hullett JB, Somera L, Barbee RW, Ward KR, Berger BE, Spiess BD. Intravenous perfluorocarbon emulsion increases nitrogen washout after venous gas emboli in rabbits. Undersea Hyperb Med. 2007 Jan-Feb; 34(1):7-20.
  12. Dromsky DM, Fahlman A, Spiess BD: Treatment of Severe decompression sickness in swine with OxygentTM, a perfluorocarbon emulsion. Aerospace Medicine 2004; 75:301-5.

Perfluorocarbon Emulsions and Platelet Activation/Sequestration

The use of Fluosol DA-20 (20% PFC), was approved by the FDA in the early 1990’s for the treatment and prevention of myocardial ischemia during the early years of coronary balloon angioplasty. It was supplied frozen and needed to be thawed and resonicated prior to usage. It was also thought to have side effects of compliment activation and potential liver enzyme activation. In second and third generation PFCs the emulsions have been stable, much like propofol, but a class-wide side effect of thrombocytopenia has been noted in approximately 30-60% of humans during phase II and III trials. In a pivotal trial of Oxygentâ„¢ (Alliance Pharmaceuticals), those patients receiving PFC had a numerically higher rate of stroke than those receiving placebo. It was hypothesized that perhaps PFC particles stimulated platelet aggregates and/or were activating platelets. The FDA has sought explanation for this class effect prior to approving further human trials. Our research group in collaboration with the Coagulation Laboratory of the VCU School of Pharmacy is in year 2 of 3 for a large animal series of studies investigating PFC platelet and white cell interactions. In year one, healthy sheep were top loaded with PFC and over 7 days the following tests were conducted: coagulation studies, platelet counts, flow cytometry, platelet aggregation, thrombo-elastography, RoTEM, platelet contractile force, platelet mean corpuscular volume, and scanning electron microscopy. The data from that top load series of studies, using two different PFCs compared to saline or hetastarch, are being evaluated now. In year 2 the sheep underwent one hour of hemorrhagic shock to a mean BP of 40 mm Hg, were partially resuscitated with hetastarch and then received PFC or saline. The same coagulation and platelet indices were studied as in year 1. In year three the model will have added to it further complexity of injury. Animals will be anesthetized and subjected to blast-induced traumatic brain injury (TBI) followed by secondary hemorrhagic shock and partial resuscitation with hetastarch. Then they will be treated randomly with PFC or saline. The same coagulation and platelet indices will be studied as in year 1. The 3-year goal of this series of experiments is to: 1) explore mechanisms of PFC platelet activation/thrombo-cytopenia generation, 2) explore whether the coagulopathy of shock is worsened by the presence of PFC in resuscitation and 3) explore the complex model of closed head injury and hemorrhage (a battlefield model) as a “worst case scenario.” We believe that if PFC infusion does not create a worsening or additive coagulopathic dysfunction, then it would be reasonable to start Phase II human trials with focused hematology studies regarding the platelet and white cell issues. To date we have presented abstracts at the annual Military Health System Research Symposium but no publications in the literature from this work.

Grants:

Sponsor: U S DoD, US Army Combat Casualty and Materiel Command, Ft Detrick, MD. Grant # W81XWH-13-1-0017, VCU # PT107803
Title: Investigations into the Extent and Mechanisms of Thrombocytopenia with Perfluorocarbon Emulsions.
Dates: May 2013 - April 30, 2016 (3 Year award).
Costs: Total Award: $2,994,479.00.

PFC and Traumatic Brain Injury Research

This collaborative multi-year series of projects has involved the Departments of Anesthesiology and Neurosurgery at VCU and the Department of Neurosurgery at the University of Miami, Ross Bullock, MD. PFC emulsions enhance O2 diffusion from red blood cells to tissues in the microcirculation as shown by our group. Traumatic, closed-head concussive injury causes initial neuron/axon shearing and cellular damage. That first wave of cell destruction is followed by hours to days of secondary inflammatory cascading events including cellular swelling, calcium metabolism dysfunction, microcirculation dysregulation, reduced blood flow, penumbral advancement and cellular apoptosis. Prior clinical and animal studies have demonstrated that early restoration of at-risk brain tissue O2 partial pressure to near 20mmHg salvages brain tissue. Hyperbaric treatment in animal models has improved neuron survival, but it is impractical for wide scale human trauma and battlefield head trauma utilization. The work by our group using PFCs tested the hypothesis that early intravenous infusion with PFC would improve at-risk neurologic tissue PtO2, preserve sensitive brain cells and improve outcomes. In several rat fluid percussion studies and sub-dural hematoma TBI studies the use of post-injury PFC infusion increased brain tissue oxygenation and improved mitochondrial survival. In a weight drop TBI study, post-injury PFC infusion also conserved axonal integrity and improved neurological outcome as measured histologically. At VCU Medical Center a human pilot trial of 9 severely head-injured patients (Glasgow comma <10) was carried out. Brain Licox probes and micro-dialysis catheters were implanted for management and measurement during the recovery phase. These patients all received PFC within 12 hours of injury and/or decompressive craniectomy. Data were compared to historical controls. The brain tissue O2 levels increased quickly after PFC infusion and the biochemical measures of tissue injury, such as lactate-pyruvate ratios and brain glutamate, improved with PFC usage as compared to historical controls. In support of our prior work, it did not matter whether these patients breathed 50% FiO2 or 100% FiO2, the biochemistry and outcomes looked the same. Further human trials were proposed but are pending the completion of our platelet projects. Our neurological research group, led by J. Travis Parsons, PhD, VCU Neurosurgery, has also had designed and custom built, in collaboration with Dyn-Fx (expert blast physicists), ORA, Inc. (mechanical engineers) and Custom Design and Fabrication South (metal fabricators), an advanced laboratory blast simulator (ABS). This type of ABS, one of only 9 similar units in the world, is emerging as the standard model of laboratory blast. It is capable of simulating exposure to free blast overpressure waves in small animals (whole body or head isolated). Blast-induced TBI is the signature injury in military conflicts of the past 2 decades in Southwest Asia. Mild to severe and repetitive TBI are high priority research areas for the military. The new ABS is currently being characterized for blast overpressure wave physics and new animal injury models are being developed. Once operable, the ABS will provide us the capabilities of developing new models, increasing our knowledge of basic blast science and designing treatment-based TBI research protocols. Previous ONR funded studies on single and repetitive air-blast-induced TBI used an older, now outdated, blast model. That work led to the fabrication of the ABS and tested the hypothesis that PFC given shortly after exposure to blast would improve outcomes. Data revealed that PFC treatment decreased the expression of biomarkers of neuronal necrosis/apoptosis (alpha II spectrin breakdown products) and improved Morris water maze cognitive performance compared to saline treatment in rats. Data from these studies have been presented at the Society for Neuroscience, Military Health System Research Symposium, and ONR Force Health Protection Future Naval Capabilities Review meeting. Further proposed research into the mechanisms of neurologic protection/salvage by PFCs are in submission and in planning.

Grants:

1. Sponsor: Office of Naval Research ONR#N000141010675. VCU # PT105710.
Title: A Dual Large and Small animal Study of the Treatment of Traumatic Brain Injury from Repetitive Blast Exposure with Oxycyte, a Perfluorocarbon Based Oxygen Therapeutic Emulsion. PI: J. Travis Parsons, PhD, Co-Investigator: B. D. Spiess, MD.
Dates: 04/01/2010 - 03/31/2013.
Costs: Total Direct: $1,775,117.00. Total F&A: $837,598.00. Total Grant: $2,612,715.00.

2. Sponsor: Synthetic Blood International, Inc./Oxygen Biotherapeutics, Inc., Costa Mesa, CA.
Title: Studies in the Basic Science of Oxygen Delivery with Oxycyte. PI: B. D. Spiess, MD.
Dates: 09/01/2008 - 09/01/2009.
Costs: Total Grant: $1,029,000.00.

3. Sponsor: Office of Naval Research ONR #N000140810366.
Title: Programmatic Multidisciplinary Studies into the Mechanisms and Efficacy of Treatment of Traumatic Brain Injury with Perfluorocarbon Oxygen Therapeutic Emulsion.
Dates: 02/2008 - 03/2009.
Direct Cost: $299,735.00. Indirect Cost: $90,349.00.
Description: This award is for the cooperation with Luna Innovations, LLC, for Embolism Detection and Classification Device.

4. Sponsor: U S DoD, Department of the Army. Grant # N000140810462.
Title: Clinical Phase II-B Trial of Oxycyte Perfluorocarbon in Severe Human Traumatic Head Injury. Co-PIs: Bruce D. Spiess, MD and M. Ross Bullock, MD, Professor of Neurosurgery, University of Miami.
Dates: 02/2008 - 03/01/2011.
Costs: $1,900,000.00. Sub-Award: $600,000.00. to B D Spiess, MD, for Trial at VCU Medical Center.

References:

  1. Daugherty WP, Levasseur JE, Sun D, Spiess BD, Bullock MR. Perfluorocarbon emulsion improves oxygenation and mitochondrial function after fluid percussion brain injury in rats. J Neurosurg 2004; 54:1223-30.
  2. Kwon TH, Sun D, Daughter WP, Spiess BD, Bullock MR. Effect of perfluorocarbons on brain oxygenation and ischemic damage in an acute subdural hematoma model in rats. J Neurosurg. 2005 Oct; 103(4):724-30.
  3. Maluf DG, Mas VR, Yanek K, Sone JJ, Weis R, Massey D, Spiess BD, Posner MP, Fisher RA, Molecular markers in stored kidney using perfluorocarbon-based preservative solution preliminary results. Transplant Proc. 2006:38; 1243-6.

Coagulation Function and Major Trauma

In conjunction with the VCUMC School of Pharmacy, Departments of Surgery (Trauma, Research) and Medicine, a series of animal and human studies are coordinated and focused upon trauma induced coagulopathy. Recognized for its novel research based coagulation laboratories, VCU’s dedicated multidisciplinary research teams are studying hemorrhagic shock in animal studies (large swine and rabbit) with and without tissue injury (bone and liver crush). A hemorrhagic shock model was developed in the coagulation laboratory and is being utilized for PFC sheep experiments. In parallel, a human prospective study of patients with trauma induced hemorrhagic shock, sponsored by the US Army to examine platelet levels and function, is completed and in manuscript review and revision. This study revealed many different coagulation measures changing in different directions at once with no single measure identified as best to follow. Additionally, the insult of a closed head injury, coupled with hemorrhagic shock, is found substantially to drive platelet numbers and function as well as the complexity of coagulopathy. In response to literature, arising from the Iraq and Afghanistan military conflicts, supporting massive transfusion protocols, a study by the United States Military hypothesizes that early intervention with plasma may decrease trauma induced coagulopathy. VCU received a 3-year, 5.6 million dollar grant for a human trial of early (in field) intervention with human plasma compared to normal saline. The VCU trauma study is part of a 3-institution collaboration with the University of Pittsburgh and the University of Colorado. Studies at each site are conducted independently of one another but in parallel, with each having similar but different outreach EMT protocols. They are utilizing human plasma and different samples collected at different time points for coagulation research. This VCU research project is approved by the FDA, VCU IRB, and DoD and coordinated with two Emergency Response Agencies (Richmond Ambulance Authority and Henrico County Fire Division). It is expected to enroll up to 200 patients with major traumatic hemorrhage over the next 3 years. An in-depth study of coagulation will occur on blood samples drawn at given time points, i.e., at time of first EMS contact, on arrival to the emergency department, and at 8 and 24 hours after arrival. Thirty-day mortality is the major outcome point and many secondary outcomes are being followed as well. As a follow-up to the plasma study discussed, a collaborative NIH (NHLBI) and US Army study will receive frozen de-identified plasma from these three Universities. Eleven to 17 different basic science laboratories around the country will request plasma from severely injured trauma patients with large amounts of de-identified demographic, hospital course and outcome data. This 2000-plus patient study is named the Trans-Agency Collaboration for Trauma Induced Coagulopathy (TACTIC). The VCU research staff is identifying the mechanisms necessary to supply samples and data to TACTIC. Our research group has official notice of award by the US Army for a 2-year, large animal study of trauma and hemorrhagic shock- induced coagulopathy and the intervention of high dose intravenous vitamin C. Vitamin C is a profound anti-oxidant. It is quickly consumed during trauma and some early work in sepsis has shown that pharmaceutical dosages have biologic activity on reducing inflammation, as well as perhaps ischemia and reperfusion injury. Work here will rapidly translate from bench top to a human trial or series of trials. Dedication to research on coagulopathy, biomarkers and treatment strategies for trauma and hemorrhagic shock form a major part of our focus. A large multi-disciplinary group at VCU has submitted a P-50 center grant to study many of these subjects (hypothesis driven) and wound healing in trauma/shock patients.

Grants Awarded:
1. Sponsor: Department of the Army Combat Casualty Care and Materiel Command, Ft Detrick.
Title: Pre-Hospital Use of Plasma in Traumatic Hemorrhage. PI: B. D. Spiess, MD
Dates: 3-year award: June 2012-June 30-2015.
Total Award: $5,572,494.00.

A novel pharmaceutical treatment for carbon monoxide treatment utilizing a unique reduced vitamin B-12 analogue.

Carbon monoxide poisoning is the most common toxic substance responsible for emergency room visits and patient deaths in the US (15,000 ED visits and 5,000 deaths) and world-wide. It is the most common method of suicide. Although not often followed in smoke inhalation (up to 600,000 cases per year in the US), it is the leading cause of toxicity from smoke inhalation. In anesthesiology, rare CO poisoning from dysfunctional CO2 reabsorption is possible. CO is now being investigated as a trace gas cell signaler akin to NO, together known now as gaso-transmitters. Small amounts of CO may be protective in terms of down regulating apoptosis. Historically, medicine has believed, since Haldane, that CO causes its toxicity by competitively binding to hemoglobin and blocking out the ability for hemoglobin to function in oxygen transport. Considerable data are contrary to that theory yet the only therapy utilized today for CO poisoning is high FiO2 and even hyperbaric oxygen. Work from the 1960s-70s suggested that, at least theoretically, the corrin ring containing cobalt of vitamin B-12 might well catalyze a reaction of CO to CO2. That work went dormant for 40 years as the laboratory methods of the 1960s-70s were simply not sophisticated enough to be able to test the hypothesis. Over the last 3 years a multi-disciplinary group of investigators from the VCU Departments of Anesthesiology, Chemistry, Burns/Plastic Surgery, Neurosurgery, Emergency Department, Toxicology, School of Pharmacy and the School of Engineering have been working on a series of projects testing a series of hypotheses regarding reduced vitamin B-12 and the detoxification of CO from hemoglobin and other tissues. In a bench top, ex vivo artificial circulation model it has been shown that our reduced B-12 agent, when added to warm circulating fresh human whole blood, displaces CO and releases CO2 in the molar quantities predicted by the early biochemists’ equations. In our laboratories, in conjunction with the Department of Chemistry, unique Raman spectroscopy techniques were invented to measure carboxy-hemoglobin accurately as well as to quantify the antidote present in circulating blood. Rats exposed to CO and treated with antidote underwent behavioral testing and histology investigations of brain injury. Some improvement was shown in animals treated with antidote v. placebo. In another set of rat studies, using implanted brain Licox probes looking at real time brain tissue oxygen levels with CO poisoning, our group was able to demonstrate that brain levels of decreased tissue oxygenation may not be directly correlated to carboxy-Hgb levels. Furthermore, when antidote was given, the brain oxygen levels rose and improved quickly towards baseline. This research has led to multiple lines of research including: drug-development, drug administration development, monitoring technology development, small and large animal trials in preparation (3 year time line) for human trials and basic science research regarding CO mechanisms of toxicity such as free radical lipid toxicity, other iron-based metaloproteins-cytochromes and others.

Reference:
Roderique JD, Van dyke K, Homan B, Tang D, Chui B, Spiess BD. The use of high-dose hydroxocobolamin for vasoplegia syndrome. AnnThorac Surg 2014; 97: 1785-6. PMID: 24792267.