USUHS Anesthesiology Disclaimer
These web pages are maintained by: Frank Olszewski.
E-mail suggestions about this website to: folszewski@usuhs.mil
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Neurotrauma Research Laboratory |
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Background The USUHS Division of Critical Care Medicine (CCM) and the Neurotrauma Research Laboratory (NTRL) are located within the Department of Anesthesiology at the Uniformed Services University of the Health Sciences (USUHS). The Division of Critical Care Medicine (CCM) was established in 1995, at the same time as the Neurotrauma Research Laboratory (NTRL). The purpose of the CCM and NTRL is to provide an academic center for critical care medicine within the military medical community. The objective of the CCM is to be the primary academic military resource for education and research as it pertains to critical care medicine. The primary research focus of the CCM and NTRL is traumatic injury to the central nervous system (CNS) – brain and spinal cord. Research is conducted at the basic science, applied science and clinical science levels. The research is focused on developing improved diagnostic and treatment strategies to managing neurotrauma. Neurotrauma is a significant health problem in both the military and civilian sectors. In the civilian sector, close to 8 million patients each year are evaluated for head injury. Close to 500,000 are hospitalized of which 100,00 die. In the military, neurotrauma accounts for over 20% of battle related casualties. A crucially important fact is that neurotrauma accounts for close to 50% of patients who die after reaching medical care. Clinical management of neurotrauma needs improvement. At present, most therapy is supportive. The lack of specific effective therapy is largely due to an incomplete understanding of this disease process. Furthermore, the ability to deliver care is hindered by lack of proper diagnostic tools for first responders – emergency medical technicians (EMT) or medics. The NTRL is dedicated to exploring all of these facets of neurotrauma. Ongoing research protocols are as follows: Cytokine Regulation of Neuroinflammation The effect of AMPA Antagonism on Traumatic Brain Injury in Rats Physiologic Tolerance Levels Following Traumatic Brain Injury The Role of Leukotrienes in Regulating Edema
A. Multipurpose RF Triage Tool Back to Top Investigators: Principal Geoffrey S.F. Ling, M.D., Ph.D., LTC, MC, USA Associate Professor and Director, Critical Care Medicine, USUHS Associate Jeremy Blanchard, M.D., MAJ, MC, USA Deputy Director, Critical Care Medicine, USUHS
Michael K. Rosner, M.D., CPT, MC, USA
Neurotrauma Resident, University of Maryland Shock Trauma Center Funding: Center for Innovative and Minimally Invasive Technology, Boston, MA (renewed for 2nd year of funding) Research Objectives:
Methods: Our study uses a hand held antenna of unique design with an operating range of 2 to 6 GHz. Illumination and detection are both accomplished via the same antenna, and the amplitude of the signal is recorded and analyzed. Electromagnetic waves in the RF ranges electrically interrogate tissues using the multiple signal classification algorithm (MUSIC). MUSIC was previously developed for estimating the angle of arrival of radar signals. The recorded signal following interrogation is a function of the scatter pattern and permittivity of the interrogated tissue. Raw data is collected using a network analyzer. Data is processed using the MUSIC algorithm and the MATLAB computer program. The data is displayed as amplitude or phase as a function of radio frequency. Both real and imaginary data are used. Investigators analyzing the raw data are blinded to the treatment. For intracranial hemorrhage studies, male Yorkshire pigs weighing from 25-50 kg are used. Each pig is premedicated with ketamine and xyloxine, I.M.. Following, pentobarbital, I.V., is used as an anesthetic. A catheter is placed surgically through a burr hole into the epidural, subdural, intraparenchymal or ventricular space. Interrogation using the RF device is performed prior to introduction of blood so that each animal can serve as its own control. Subsequently, autologous blood is administered via the catheter into the appropriate space. Shortly after treatment, each pig is again interrogated using the RF device. The antenna is held 6 inches away from the pig’s head. For pneumothorax and compartment syndrome studies, male Yorkshire pigs are again used. Each pig is premedicated and anesthetized as above. For pneumothorax experiments, a catheter is placed surgically into the pleural space. Increasing volumes of air are instilled. For compartment syndrome experiments, a catheter is placed into the muscle compartment of a hind limb. Increasing volumes of saline or blood is instilled. Interrogation using the RF device is performed prior to introduction of air, saline or blood so that each animal can serve as its own control. Subsequently, after administration, each pig is again interrogated using the RF device. The antenna is held 6 inches away from the pig. Human studies will also be performed. Normal volunteers will be studied to determine baseline signature characteristics. Each subject will be undergo interrogation of the head, chest and leg. Data will be collected and will serve as baseline control data for subsequent future studies in patients. Outcomes: Our work confirms the hypothesis that RF imaging can be applied to noninvasively determine the presence or absence of intracranial hemorrhage. The RF device can differentiate the presence of as little as 2cc of subdural blood from baseline. It can reproducibly and reliably determine as little as 5cc of epidural, intraparenchymal and intraventricular blood from baseline. Preliminary work demonstrates the ability of the RF device in identifying 100cc of air in the pleural space. Further work is being done to complete in vivo validation of the RF device as a noninvasive portable tool for diagnosing intracranial hemorrhage, pneumothorax and compartment syndrome. All of these are common combat casualties. After which, we will begin human trials in appropriate patients.
B. Cytokine Regulation of Neuroinflammation Back to Top Investigators: Principal Geoffrey S.F. Ling, M.D., Ph.D., LTC, MC, USA Associate Professor and Director, Critical Care Medicine, USUHS Associate Jeremy Blanchard, M.D., MAJ, MC, USA Deputy Director, Critical Care Medicine, USUHS Funding:
Research Objectives
Methods: To begin, we developed a novel animal model that allows us to examine the effect of cytokines within the closed system of the CNS. It is critical that the protective covering of the CNS, i.e., blood brain barrier (BBB), remain intact. Breeches in the BBB allow influx of inflammatory mediators (cytokines, chemokines, white blood cells, etc) from blood, which would contaminate results. Also, as the BBB is living tissue, acute injury from a needle puncture may incite inflammation, which would also contaminate results. Our model is based on implantation of a chronic intracerebroventricular catheter attached to an Ommaya reservoir. After 1 month of healing to reestablish the integrity of the BBB, the apparatus allows cerebrospinal fluid (CSF) sampling and exogenous cytokine administration without acute breech of the BBB. Using this approach in a canine model, we recreate the release of cytokines that would occur after traumatic injury. In this way, we are able to reproduce the initiating events of inflammation without having to traumatize the animal. Following, we follow the expression of a commonly used clinical marker of inflammation in CSF, i.e., white blood cell count. Outcomes: Our work demonstrates that IL-1 causes a very early CSF leukocytosis that is primarily lymphocytic in nature. This begins at 10 minutes after dosing, peaks within 1 hour and then persists for hours. Pharmacokinetic analysis of IL-1 in the CSF reveals an elimination T1/2 of 45 min. This is much too rapid for sustained IL-1 levels to be the basis for the prolonged inflammatory state. Further work revealed that TNF is released in response to IL-1 and persists. IL-6 is not. Thus, the inflammatory state induced by IL-1 is, in part, mediated by TNF. Studies on TNF reveal a delayed predominately neutrophilic cellular response. This effect begins at approximately 1 hour and does not peak until close to 4 hours. TNF elimination is very rapid, approximately 20 minutes. In response to TNF, neither IL-1 nor IL-6 is released. Thus, the mediation of the prolonged neuroinflammatory state induced by TNF is sustained by another, as of yet, unidentified cytokine. IL-6 studies also reveal a delayed cellular response, which is predominately lymphocytic. Onset begins at 45 minutes but peak does not occur until 10 hours. Pharmacokinetic analysis of IL-6 reveals a T1/2 of approximately 20 minutes. Neither IL-1 nor TNF is released in response to IL-6. Again, mediators of IL-6’s prolonged effect need to be identified. Our work demonstrates that the brain has remarkable inflammatory capacity. The neuroinflammatory state of trauma is largely lymphocytic. Thus, it is likely initiated by IL-1 release which in turn causes TNF release. IL-6 is also likely released early but its effects are delayed. If specific anticytokine therapies are to be used to treat neurotrauma, then it will be important to tailor drug regimens to specific treatment times after injury. The goal should be to target the cytokines most active at that time. Thus, treating with an anti-IL-1 agent should be as early as possible but anti-TNF treatment should be delayed at least 1 hour.
C. Tool for Combat Corpsman Use to Identify and Triage Traumatic Brain Injury in the Far Forward Environment Back to Top Investigators: Principal Geoffrey S.F. Ling, M.D., Ph.D., LTC, MC, USA Associate Professor and Director, Critical Care Medicine, USUHS Associate Jeremy Blanchard, M.D., MAJ, MC, USA Deputy Director, Critical Care Medicine, USUHS
Michael K. Rosner, M.D., CPT, MC, USA
Funding Status: Combat Casualty Care program, Office Naval Research, 1-year grant Research Objectives:
Methods The clinical tool will be based on skull integrity, seizure, level of consciousness, pupillary function, and pattern of weakness. In addition to this tool, every aspect of the traditional neurologic examination (i.e., speech, cranial nerve function, visual field analysis, sensory, deep tendon reflexes, muscle tone, coordination and gait) will be measured to identify any other predictive factors for traumatic brain injury. An initial retrospective review of multiple trauma charts will be reviewed to establish validity of the clinical tool and determine if other aspects of the neurologic examination are useful in diagnosing brain injury. A subsequent prospective trial will then be performed applying the refined clinical tool to incoming trauma patients. Multiple charts will be reviewed of trauma patients with head injuries who presented to a local Level 3-trauma center. Findings from the complete neurologic and general physical examination on presentation to the trauma center will identified and annotated for a potential predictive finding in head injury. The charts will then be reviewed for the diagnosis of traumatic brain injury. Statistical analysis will then be applied to initial findings on exam and diagnosis of brain injury. A comparison will be made to the traditional approach. From this, the clinical tool will be refined. Statistically, greater sensitivity will be accepted at the expense of specificity. In other words, the clinical tool will be developed with the intention of identifying all brain-injured patients (high sensitivity) with the acceptance of a higher number of "false" positives (lower specificity). Incoming trauma patients who present to a separate local Level 3-trauma center will be evaluated using the clinical tool developed from the data obtained in the retrospective study. During emergency department evaluation, a physician examiner blinded to the patient's final diagnosis will use the tool to examine each patient with suspected brain injury. The patient will receive appropriate medical management based on evaluation by the primary treating physician. Thus, this study will have no impact on application of standard of care and will not be used in triage, surgical or medical management decision making or prognosis. The final diagnosis regarding traumatic brain injury will be recorded after completion of patient evaluation and care. Statistical analysis and significance will be applied to the data comparing the reliability of the clinical tool to traditional approach in identifying brain injury. Outcomes: Ongoing
D. The effect of AMPA Antagonism on Traumatic Brain Injury in Rats Back to Top Investigators: Principal Geoffrey S.F. Ling, M.D., Ph.D., LTC, MC, USA Associate Professor and Director, Critical Care Medicine, USUHS Associate Richard McCarron, Ph.D. Director, Resuscitation Laboratory, NMRI Michael Rogawski, M.D., Ph.D., Director, Epilepsy Branch, NIH Michael K. Rosner, M.D., CPT, MC, USA
Funding status: Hemorrhagic Shock and Resuscitation program, Office of Naval Research, 1-year grant Research Objectives:
Methods: Rats will be anesthetized with N2O, halothane and oxygen mixture. A small burr hole will be made in the skull positioned parasagittally over the right cerebral cortex. A canula will be introduced through the hole until it abuts the pia surface. Sprague-Dawley rats will be used. Fasted rats will be anesthetized with 69% N20, 1% halothane, and 30% oxygen and mechanically ventilated. Under controlled physiological conditions and normothermic brain temperature, TBI will be induced by an established model of closed head injury, the fluid percussion device. In brief, a continuous fluid column is established between the surface of the rat brain and a fluid filled piston. A pendulum arm is used to strike the piston, which creates a fluid wave. The wave is propagated along the fluid column until it strikes the rat’s brain. This is the basis of the injury. LY293558, a novel AMPA receptor antagonist, is administered i.v. 15 minutes after injury. The dose is 10 mg/kg. Rats are monitored continuously for 6 hours after injury. Neurobehavior is measured using the McIntosh Behavior Score and the NIH Stroke Scale. Measurements are made at 1, 24, 28 and 72 hours after TBI. At 72 hours, animals are sacrificed and the brains are removed. Histopathology quantitation of injury is made on each animal’s brain. Brain sections are stained with H&E to allow determination of lesion size. Necrosis is quantitated by counting dead neurons. Results are mean number of dead neurons in 3 different high powered fields per brain region. Brains are also stained with TUNL. Apoptosis is quantitated by counting TUNL stained cells. Results are mean values of 3 different high-powered fields per brain region.
Outcomes: The results of the present study demonstrate a neuroprotective effect when LY293558 is given 15 minutes after traumatic brain injury in the fluid percussion model of brain injury in a rat. The neuroprotective effect is demonstrated not only in neurobehavioral evaluation but also histopathologic analysis to include H&E, cresyl violet and TUNEL staining The quoted mortality rate for a level of injury at 2.5 ATM, which is considered a moderate/severe level of injury, is approximately 20%. In our control group, the mortality rate was 24% (n =17). The mortality rate for the drug treated group was 0% (n =11). LY293558 treated rats had faster recovery than control rats. Furthermore, all reached full recovery by 48 hours whereas untreated rats still had residual deficits at 72 hours. The clinical grading of the treated rats at 24 and 48 hours demonstrated significantly improvement (p < 0.05). The neurobehavioral grading of the treated rats at 24 and 48 hours also demonstrated significant improvement (p < 0.05). H&E and cresyl violet stains of rat brains at the impact site demonstrated less surrounding brain edema and neuronal death of the drug group compared to the control group. The impact site in both groups demonstrated subarachnoid and intraparenchymal hemorrhage consistent with primary injury. The secondary injury of surrounding edema was decreased in the drug group. The improvement was seen at higher power magnifications at the margin of primary injury and surrounding edema. The level of apoptosis was evaluated with TUNEL staining. Coronal slices through the center of impact site were prepared as described above. Six regions were evaluated with manual counting of the positive staining cells. A decrease in apoptotic cells was noted in all regions (p < 0.05) with the most profound sites being the ipsilateral and contralateral hippocampal regions.
E. Physiologic Tolerance Levels
Following Traumatic Brain Injury
Back to Top Investigators: Principal: Jeremy Blanchard, M.D., MAJ, MC, USA Assistant Professor and Deputy Director, Critical Care Medicine, USUHS Associate: Geoffrey S.F. Ling, M.D., Ph.D., LTC, MC, USA Associate Professor and Director, Critical Care Medicine, USUHS Funding Status: Under review by USAMRMC Research Objectives: The overall long-range objective of this study is to develop a rational approach for clinical management of combat related traumatic brain injury (TBI). The intended purpose of this research is to provide basis for developing treatment and triage algorithms to optimize neurologic, functional and survival outcome of soldiers with TBI when resources such as supplemental oxygen, blood or i.v. fluids are limited.
Methods: TBI will be produced as described above. For evaluation of the effects of different levels of hematocrit the animals are anesthetized with general (isoflurane) anesthesia. A femoral arterial line is placed using a cut down technique. The arterial line allows controlled bleeding as well as arterial blood sampling and establishment of a baseline hematocrit (the "normal" hematocrit of a male rat is 40). At regular intervals sampling of arterial blood gases allow for FIO2 adjustment and minute ventilation adjustment to keep their oxygen level greater than 100 mmHg, and their PCO2 between 30 and 40 mmHg. After baseline physiologic measurements have been made, the rat is prepared for fluid percussion TBI as described above. Each rat then receives a 2.5 atm fluid percussion pulse to their cerebrum. The animals are then randomized to one of five levels of percent hematocrit-40, 35, 30, 25 and 20. Blood is removed using a model of uncontrolled hemorrhage that was previously described 15. After one hour at the specified hematocrit, the catheter removed. The animal is then extubated and returned to his cage with available water and food. For evaluation of the effects of different levels of oxygenation, the animals will be prepared as above. The animals are then randomized to one of five levels of oxygenation-PaO2 equal to 100, 80, 60, 50, and 40 mmHg. Each rat is maintained on mechanical ventilation and general anesthesia. An oxygen blender is used to adjust the FIO2 by mixing oxygen with nitrogen. After one hour of hypoxia, the catheter removed. The animal is then extubated and returned to his cage with available water and food. For evaluation of the effects of different levels of oxygenation, the rats will be prepared as above. Anesthesia will be maintained throughout as above. The animals are then randomized to one of five levels of blood carbon dioxide-PaCO2 equal to 40, 45, 50, 55 and 60 mmHg. The FIO2 and ventilator rate is adjusted to keep the PaCO2 at the indicated level. After one hour, the catheter is removed. It is then extubated and returned to his cage with available water and food. After injury, animals are allowed to recover fully from anesthesia and monitored. At 1, 24, and 72 hours the animal under goes a series of neurological tests to determine residual function and severity of injury. The animal is then sacrificed and the brain harvested per the tissue analysis method portion of this proposal. Animals behavior are evaluated using 2 neurologic severity score systems, Neurologic Severity Score and NIH Stroke Scale, over a period of 3 days, i.e. at 1, 24, 48 and 72 hours after injury. Outcome is determined by comparing NSS and NIHSS scores in TBI rats subjected to physiologic stresses as compared to those who were not. Survival is a direct comparison of the number of TBI rats surviving per group. At 72 hours after injury and neurologic scoring, animals are then reanesthetized and cardiac perfused with buffer solution. Brains are harvested and frozen. Tissue is then sectioned and stained with either H&E or TUNEL. This allows histopathologic evaluation by light microscopy. These techniques have been well characterized for TBI. Necrosis is defined by the decreased of neurons in injured brain tissue as compared to normal. Apoptosis is defined by the increased number of apoptotic cells counted in injured brain tissue as compared to normal. Outcomes: Ongoing
F. The Role of Leukotrienes in Regulating Edema Back to Top Investigators: Principal Masako Nozaki, Ph.D. Assistant Professor, Critical Care Medicine, USUHS Associate Geoffrey S.F. Ling, M.D., Ph.D., LTC, MC, USA Associate Professor and Director, Critical Care Medicine, USUHS Funding status: Proposal undergoing review by USA-MRMC Research Objectives: 1. To determine the effect of leukotriene (LT) antagonists on edema produced in an
2. To determine the effect of leukotriene antagonists on edema produced in brain Methods: To address the first objective, the rat hind paw edema test will be used. In brief, an edema producing agent is injected into the hind paw of a rat. Edema quickly develops so that by 3 hours, the full syndrome is manifested. The amount of edema produced is quantitated by the water displacement technique. Various therapeutic manipulations are then instituted in an attempt to ameliorate this condition. Specific to these studies, the edema producing agents are dextran (putatively causes leukotriene related edema), carrageenin (cyclooxygenase (CO) related edema) and arachidonic acid (both lipoxygenase and cyclooxygenase products). To determine the contribution of LT in these edema states, rats will be treated with pranlukast, a putative LT antagonist. Edema associated with traumatic brain injury will also be explored. TBI will induced as above. Pranlukast will be administered both with and without indomethacin, a cyclooxygenase inhibitor of prostaglandin synthesis. This will be performed to ascertain the efficacy of eicosinoid therapy in TBI. Outcomes: ongoing This page was last updated on July 23, 2001 |
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USUHS Anesthesiology Disclaimer These web pages are maintained by: Frank Olszewski. E-mail suggestions about this website to: folszewski@usuhs.mil |