Synergistic effects of fresh frozen plasma and valproic acid treatment in a combined model of traumatic brain injury and hemorrhagic shock

Synergistic effects of fresh frozen plasma and valproic acid treatment in a combined model of traumatic brain injury and hemorrhagic shock Ayesha M. Imam, MD,a Guang Jin, MD, PhD,a,c Michael Duggan, DVM,a Martin Sillesen, MD,a John O. Hwabejire, MD, MPH,a Cecilie H. Jepsen, MD,a Danielle DePeralta, MD,a Baoling Liu, MD,a,c Jennifer Lu, BS,a Marc A. deMoya, MD,a Simona Socrate, PhD,b and Hasan B. Alam, MD,a,c Boston and Cambridge, MA, and Ann Arbor, MI

Introduction. Traumatic brain injury (TBI) and hemorrhagic shock (HS) are major causes of traumarelated deaths and are especially lethal as a combined insult. Previously, we showed that early administration of fresh frozen plasma (FFP) decreased the size of the brain lesion and associated swelling in a swine model of combined TBI+HS. We have also shown separately that addition of valproic acid (VPA) to the resuscitation protocol attenuates inflammatory markers in the brain as well as the degree of TBI. The current study was performed to determine whether a combined FFP+VPA treatment strategy would exert a synergistic effect. Methods. Yorkshire swine (42–50 kg) were instrumented to measure hemodynamic parameters, intracranial pressure, and brain tissue oxygenation. TBI was created through a 20-mm craniotomy using a computer-controlled cortical impactor: 15-mm cylindrical tip impactor at 4 m/s velocity, 100 ms dwell time, and 12-mm penetration depth. The TBI was synchronized with the initiation of volume-controlled hemorrhage (40 ± 5% of total blood volume). After a 2-hour period of shock, animals were randomized to 1 of 3 resuscitation groups (n = 5 per group): (1) 0.9% saline (NS); (2) FFP; and (3) FFP and VPA 300 mg/kg (FFP+VPA). The resuscitative volume for FFP was equivalent to the shed blood, whereas NS was 3 times this volume. VPA treatment was started 1 hour after hemorrhage. Animals were monitored for 6 hours post-resuscitation. At this time the brains were harvested, sectioned into 5-mm slices, and stained with 2,3,5-triphenyltetrazolium chloride to quantify the lesion size (mm3) and brain swelling (percent change compared with the uninjured side). Results. The combined TBI+HS model resulted in a highly reproducible brain injury. Lesion size and brain swelling (mean value ± standard error of the mean) in the FFP+VPA group (1,459 ± 218 mm3 and 13 ± 1%, respectively) were less than the NS group (3,285 ± 131 mm3 [P < .001] and 37 ± 2% [P < .001], respectively), and the FFP alone group (2,160 ± 203 mm3 [P <.05] and 22 ± 1% [P <.001], respectively). Conclusion. In a large animal model of TBI+HS, early treatment with a combination of FFP and VPA decreases the size of brain lesion and the associated swelling. (Surgery 2013;154:388-96.) From the Department of Surgery,a Division of Trauma, Emergency Surgery and Surgical Critical Care, Massachusetts General Hospital/Harvard Medical School, Boston; the Harvard-MIT Division of Health Sciences and Technology,b Massachusetts Institute of Technology, Cambridge, MA; and the Department of Surgery,c University of Michigan Hospital, Ann Arbor, MI

Funded by a grant from the US Army Medical Research Material Command GRANTT00521959 (to HBA). Presented at the 8th Annual Academic Surgical Congress in New Orleans, Louisiana, February 2013. Accepted for publication May 10, 2013. Reprint requests: Hasan B. Alam, MD, Norman Thompson Professor of Surgery, Section Head, General Surgery, 2920 Taubman Center/5331, University of Michigan Hospital, 1500 E. Medical Center Drive, Ann Arbor, MI 48109-5331. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2013.05.008

388 SURGERY

INJURIES are the leading cause of mortality in patients under the age of 44,1 and account for >5.8 million deaths worldwide each year.2 Traumatic brain injury (TBI) occurs in 1.4 million people each year in the United States,3 and is the leading cause of disability in civilian and military settings.3,4 TBI occurs often in conjunction with hemorrhagic shock (HS).5,6 Studies have shown that the vast majority of trauma related deaths are owing to hemorrhage and TBI,7 and that HS can double the morbidity and mortality following TBI.8,9 In TBI, the extent of brain damage is determined by the initial mechanical impact (primary

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injury), as well as the subsequent insults (secondary injury). Tissue hypoxia is a well-known risk factor of secondary brain injury that contributes to increased cerebral edema, increased intracranial pressure, and more severe cellular destruction. The adverse outcomes in patients with combined TBI and HS are most likely owing to the secondary ischemic insult to the already vulnerable brain as a result of disrupted cerebral autoregulation.10,11 It is also possible that TBI could adversely affect the normal compensatory responses to HS.12,13 Therefore, it is crucial to select an appropriate strategy of resuscitation during the early management of patients with TBI, especially when TBI is complicated by the presence of HS. An ideal resuscitation strategy should replenish the lost blood while minimizing secondary brain injury. Traditionally, crystalloids have been the standard resuscitation fluid14,15; however, supporting evidence, especially in the setting of TBI, is limited. We reported previously that early resuscitation with fresh frozen plasma (FFP) decreases the size of brain lesion and associated swelling (compared with normal saline) in a large animal model of TBI+HS.16 An even more effective strategy would be to administer potent cytoprotective agents during the early post-trauma period. Several studies have established that valproic acid (VPA), an anticonvulsant and mood-stabilizing drug, protects against various lethal insults. In large doses, VPA acts as a histone deacetylase inhibitor that can correct shock-induced acetylation imbalance swiftly and increase survival in models of otherwise fatal HS17-19 and polytrauma.20 VPA has potent antiinflammatory and anti-apoptotic properties.21,22 More specifically, VPA protects the neurons against hypoxia-induced apoptotic cell death.23 We also demonstrated that addition of VPA to an artificial colloid resuscitation protocol attenuates inflammatory markers, decreases the lesion size, and minimizes brain swelling in a model of combined TBI and HS.24 It remains unknown whether these two promising treatments (FFP and VPA) could be combined to yield synergistic effects. We hypothesized that the combined treatment with FFP and VPA will attenuate brain lesion size and swelling more than FFP treatment alone. MATERIALS AND METHODS All experiments were carried out in accordance with the principles set by the Animal Welfare Act and other federal regulations and statutes relating to research involving animals. The study was in compliance with the Guide for the Care and Use of

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Laboratory Animals, Institute for Laboratory Animal Research (1996) and was approved by the institutional animal care and use committee. All procedures were performed under the supervision of a veterinarian, using aseptic techniques. Animal selection and preparation. Female Yorkshire swine (42–50 kg; Tufts Veterinary School, Grafton, Mass) were allowed to acclimate for 3 days and examined by a veterinarian to ensure good health. Food was withheld overnight before the experiment, but access to water was maintained. On the day of the experiment, animals were sedated with an intramuscular injection of 8 mg/kg Telazol (50 mg/mL tiletamine hydrochloride and 50 mg/mL zolazepam hydrochloride; Fort Dodge Animal Health, Fort Dodge, Ia) mixed with 1.5 mg of atropine. They were placed in a supine position, anesthesia was induced with 4% inhaled isoflurane in 100% oxygen and cuffed Silastic endotracheal tubes with a 7.0-mm internal diameter were placed to initiate mechanical ventilation (Narkomed-M, North American Drager, Telford, Penn). Initial ventilator settings included a tidal volume of 10 mL/kg of body weight, peak pressure of 20 cm H2O, and a respiratory rate of 10 breaths per minute to maintain an end-tidal PCO2 (mean value ± standard error of the mean) of 40 ± 2 mmHg. Isoflurane was adjusted between 1% and 3% inspiratory fraction to maintain inhaled anesthesia. Instrumentation and monitoring. The following vessels were cannulated using cut down techniques: Right femoral artery (blood pressure monitoring), left femoral vein (treatment/fluid administration), left femoral artery (hemorrhage, intraoperative laboratory draws), and right external jugular vein (pulmonary artery catheter placement). Post instrumentation for measurement of hemodynamic parameters, blood samples were drawn for the baseline time point. A lower midline laparotomy was then performed to place a cystostomy tube for measurement of urine output. Cardiac output (CO), SvO2, and core body temperature were monitored continuously (Vigilance II Monitor, Edwards Lifesciences, Irvine, Calif). Body surface area (BSA) was calculated (BSA = 0.0734 3 body weight0.656). The CO and BSA were used to calculate the cardiac index (CI) using the following formula: CI = CO/BSA. Invasive hemodynamic monitoring was performed continuously (Eagle 4000 Patient Monitor, GE Marquette, Piscataway, NJ), and blood pressure readings were recorded every 5 minutes. Endtidal CO2 was also measured throughout the experiment (V9004, SurgiVet, and Waukesha,

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Fig 1. A, Schematic of swine skull showing the location of the burr hole for the injury to the right frontal lobe. Location of the ICP monitor anterior to the bregma 1 cm and lateral 1 cm on the left side is also shown. B, The TBI device mounted on surgical table.

Wisc) along with pulse oximetry. The animal was then placed in a prone position, and the head was fixed in a stereotactic frame to prevent movement. A 20-mm burr hole was made on the right side of the skull next to the coronal and sagittal sutures over the frontal lobe to expose the dura (Fig 1, A). Bone was removed carefully so as not to disturb the dura and the underlying brain tissue. A double channel catheter for intracranial pressure (ICP) monitoring (Integra Lifesciences, Plainsboro, NJ) and brain tissue oxygenation monitoring (Licox system, Integra Lifesciences) were inserted through a bolt placed in a 2-mm burr hole on the left side of the skull, 10 mm lateral and 10 mm anterior to the bregma. Controlled cortical impact. A computercontrolled cortical impact device developed by the Department of Materials Science and Engineering, Harvard-MIT Division of Health Sciences and Technology (Massachusetts Institute of Technology) was used for these experiments (Fig 1, B).25 The impactor device assembly consisted of a voice coil linear actuator with a built-in Linear Variable Differential Transformer displacement transducer (H2W Technologies Inc, Santa Clarita, Calif) connected to a closed-loop motion control board (Galil Motion Control, Rocklin, Calif). A 15-mm, cylindrical impactor tip was mounted on an electronic motor, and the dynamics were controlled precisely to deliver 4-m/s velocity, 100 ms dwell time, and 12-mm depth penetration. The computer-controlled cortical impact device was attached to the stereotactic frame and secured in place for firing. After

impact, the burr hole was sealed with bone wax to prevent leakage of cerebrospinal fluid and to eliminate any artifacts in ICP monitoring. Hemorrhage and resuscitation protocol. The total blood volume was estimated, and 40 ± 5% of it was withdrawn through the femoral arterial catheter using a Masterflex pump, Model L/S Computerized Drive with a MF easy load II Pumphead, Model 77201-60 (Cole-Palmer, Vernon Hills, ILL). This volume-controlled hemorrhage was started concurrent with TBI at a rate of 3% total blood volume per minute. Shed blood was captured in a Terumo blood collection bag (CPDA and AS-5). Isoflurane was decreased with the onset of hypotension. Using this protocol, MAP was maintained between 30 Hg and 35 mmHg until 40% of the estimated blood volume was withdrawn in a controlled fashion. If the mean arterial pressure (MAP) dropped to <30 mmHg, hemorrhage was held briefly, and the animal was allowed to recover. Once the MAP reached 35 mmHg, hemorrhage was restarted. After hemorrhage, animals were left in shock for 120 minutes, and MAP was maintained between 30 and 35 mmHg by titrating the dose of inhaled isoflurane. The depth and duration of shock was based on a series of previous experiments, leading to the development of a reproducible, severe, but reversible brain lesion.16 Before the operation, animals were assigned to 1 of 3 resuscitation groups (n = 5 per group): (1) 0.9% sodium chloride solution (normal saline [NS]; HOSPIRA 0.9% sodium chloride irrigation, 3,000 mL) at 165 mL/min; (2) FFP at 50 mL/min; or (3) FFP at 50 mL/min plus VPA (EMD Biosciences, Inc, La Jolla, Calif) 300 mg/kg. Volumes of FFP matched the

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Statistical analysis. All data are presented as mean values ± standard error of the mean, unless mentioned otherwise. Group means were compared using 1-way analysis of variance with post hoc Bonferroni correction. Logarithmic transformation of the data was done to achieve equality of variances before conducting the analysis. GraphPad Prism version 5.00 (GraphPad Software, San Diego, Calif) software was used. Fig 2. Timeline of the TBI and HS model. The x axis shows time in minutes. OB, Observation; PR, postresuscitation; PS, post-shock.

shed blood, whereas the NS group given was 3 times this volume. The NS group was included as a reference and to serve as a bridge to previous studies. Although it is a valid experimental control, large-volume saline infusion (without red blood cells) is not clearly the clinical ‘‘standard of care’’ for patients in HS. In the FFP+VPA group, VPA treatment was started intravenously at 1 hour after hemorrhage at an infusion rate of 100 mg/kg per hour. FFP alone and FFP+VPA groups received identical volumes of fluids. Warmed fluids were infused into the femoral vein using a Masterflex pump. The timeline of the TBI and HS model is shown in Fig 2. Observation and harvest of samples. All animals were monitored under anesthesia for 6 hours after resuscitation. Physiologic parameters were measured continuously and recorded every 5 minutes. Animals were kept warm (Bair Hugger Model 505; Arizant Healthcare, Inc, Eden Prairie, Minn), and electrolytes and blood glucose levels were corrected as needed. At the end of the observation period, animals were killed by an intravenous injection of Euthasol (sodium pentobarbital [100 mg/kg]). A detailed autopsy was performed to evaluate all the thoracic and abdominal organs as well as the brain. The entire brain was removed for examination. Samples from other organs were collected for analysis at a later stage (separate study). Calculation of brain infarction and swelling. Brains were sliced into 5-mm coronal sections. To demonstrate the presence of nonviable tissue, slices were incubated in 2% 2, 3, 5-triphenyltetrazolium chloride (Sigma Chemical Co., St. Louis, Mo).26 ImageJ (National Institutes of Health, Bethesda, Md) computer-assisted image analysis software was used to measure the size of the lesion. Brain swelling was calculated by comparing it to the uninjured hemisphere {([ipsilateral hemisphere’s volume/contralateral hemisphere’s volume] 1) 3 100}.27 True infarction volumes were corrected by the swelling factor.28,29

RESULTS Lethality of the insult. In the current experiment, all animals survived until the end of the observation period. Hemodynamic data. Fig 3, A represents the MAP data. The baseline pressures between the 3 groups were similar (NS, 73 ± 12 mmHg; FFP, 71 ± 9 mmHg; and FFP+VPA, 70 ± 4 mmHg). An equal degree of hypotension was observed in all 3 groups after the volume-controlled hemorrhage, which persisted throughout the 2-hour shock period. During the early hours of observation, there was a difference in the MAP between the FFP and FFP+VPA groups, with the latter having a lesser MAP (observed at 2 hours, 60 ± 7 vs 41 ± 2 mmHg [P < .05]; observed at 3 hours, 57 ± 5 vs 43 ± 3 mmHg [P < .05]; observed at 4 hours, 59 ± 7 vs 46 ± 4 mmHg [P < .05], respectively). This difference was no longer at the end of the experiment. The changes in heart rate were identical in all the groups (Fig 3, B). The CI decreased after hemorrhage in all the groups (Fig 3, C) and improved to near baseline levels after resuscitation with a greater increase in the NS group (NS, 7.9 ± 1.8 L/min; FFP, 5.4 ± 0.7 L/ min; FFP+VPA, 4.6 ± 0.8 L/min; NS vs FFP, P < .05; NS vs FFP+VPA, P < .05). At the end of the 6-hour observation period, CI was greater in the FFP+VPA group when compared with the FFP alone group (P < .05). Central venous pressures decreased to the same degree in all the groups after hemorrhage (Fig 3, D). A sustained increase in the central venous pressure was not noted in any group, suggesting a volume overload. Physiologic data. The Table shows the results of the serial hemoglobin levels, pH, and serum lactate levels that were used to measure metabolic acidosis during the experiment. No differences were observed in these parameters between any of the groups until resuscitation. Immediately postresuscitation, lactate levels were greater in the FFP group as compared with the NS group (FFP, 7.0 ± 4.7 mmol/L; NS, 3.2 ± 1.2 mmol/L; P < .05). Similarly, differences occurred in the FFP+VPA group in the first 2 hours post-resuscitation when compared

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Fig 3. Mean arterial pressure (MAP; A), heart rate (B), cardiac index (CI; C), and central venous pressure (CVP; D) at selected times during the experiment. The x axis is time in minutes from initiation of TBI. *P < .05 FFP versus FFP+VPA; #P < .05 NS versus FFP+VPA; sP < .05 NS vs FFP. FFP, Fresh frozen plasma; NS, 0.9% normal saline; VPA, valproic acid.

with the NS group. Lactic acidosis improved gradually during the observation period, with the greatest degree of lactic acidosis seen in the FFP+VPA group (FFP+VPA, 3.7 ± 1.00 mmol/L vs FFP, alone, 2.6 ± 1.0 mmol/L). There was no difference in the arterial pH between the 3 groups. A transient decrease in hemoglobin level was seen in the animals receiving crystalloids immediately after resuscitation. The degree of anemia was similar between the groups at the end of the observation period. No differences were found in the ICP and partial pressure of oxygen in brain tissue measurements between the groups (Fig 4). Brain swelling and lesion size. As shown in Fig 5, FFP administration was associated with (P < .05) smaller lesion size and less swelling compared with the NS group (2,160 ± 203 mm3 and 22 ± 1% vs 3,285 ± 131 mm3 and 37 ± 2%, in the FFP and NS groups, respectively). The greatest decrease in lesion size and swelling was found in the group treated with FFP+VPA (1,459 ± 218 mm3 [P < .05] and 13 ± 1% [P < .05]). DISCUSSION In a clinically relevant large animal model of HS+TBI, we found that administration of VPA

treatment within 1 hour of insult decreased the size of the brain lesion as well as associated swelling. This study builds on our previous work16,24 by demonstrating that 2 neuroprotective treatments (FFP and VPA) can be combined to yield synergistic results. Resuscitation plays a critical role in restoring and maintaining systemic as well as the cerebral perfusion in patients with TBI. Traditional treatment protocols have advocated rapid infusion of large volumes of crystalloids in TBI patients to restore intravascular volume and correct hypotension.30,31 At the same time, laboratory and clinical research has shown that such aggressive strategies could have detrimental effects, including aggravation of cerebral edema, intracranial hypertension, and decreased brain compliance.32,33 One potential way to minimize the volume of fluids would be to use colloids for resuscitation. However, the use of colloids in the setting of TBI is controversial. The SAFE trial reported a greater mortality in TBI patients who were resuscitated with an albuminbased fluid.14 Although the precise reason behind worse outcomes remain unknown, dilution coagulopathy has been postulated as a possible

Time points Parameters pH

Lactate (mmol/L)

Hg (g/dL)

Groups

Baseline

PS

2 hr PS

PR

1 hr OB

2 hr OB

3 hr OB

4 hr OB

5 hr OB

6 hr OB

NS FFP FFP+VPA NS

7.43 7.46 7.47 1.6

± ± ± ±

0.03 0.03 0.02 0.34

7.39 7.39 7.45 3.1

± ± ± ±

0.05 0.06 0.02 1.60

7.41 7.39 7.43 3.2

± ± ± ±

0.05 0.08 0.01 1.12

7.32 7.37 7.39 3.2

± ± ± ±

0.03 0.07 0.03 1.17

7.39 7.46 7.41 2.5

± ± ± ±

0.02 0.05 0.03 0.58

7.41 7.51 7.45 2.2

± ± ± ±

0.04 0.06 0.04 0.62

7.42 7.51 7.49 1.6

± ± ± ±

0.03 0.03 0.03 0.65

7.42 7.51 7.49 1.6

± ± ± ±

0.05 0.06 0.04 0.58

7.42 7.52 7.49 1.6

± ± ± ±

0.04 0.04 0.02 0.64

7.43 7.50 7.48 1.5

± ± ± ±

0.04 0.05 0.02 0.64

FFP FFP+VPA NS FFP FFP+VPA

1.7 1.3 9.4 9.5 9.8

± ± ± ± ±

0.37 0.30 1.17 0.61 0.35

3.6 2.2 10.7 10.4 10.8

± ± ± ± ±

1.23 0.70 0.77 0.73 0.66

6.3 3.9 10.1 10.4 11.0

± ± ± ± ±

4.31 0.96 1.03 0.90 0.67

7.0 5.5 4.9 8.3 8.4

± ± ± ± ±

4.73* 1.77 0.80 0.89* 0.53*

5.5 6.6 5.7 7.6 7.5

± ± ± ± ±

4.27 2.20* 0.65 1.10* 0.77*

4.1 6.3 6.6 6.4 6.9

± ± ± ± ±

3.73 2.52* 0.47 1.10 0.76

4.0 4.9 6.7 6.2 6.7

± ± ± ± ±

2.88 1.73 0.71 0.79 0.68

3.6 3.9 7.1 6.3 6.8

± ± ± ± ±

2.00 1.53 0.57 0.62 0.75

2.9 3.4 7.0 5.8 6.7

± ± ± ± ±

1.18 1.38 0.76 1.07 0.35

2.6 3.7 6.6 6.6 6.6

± ± ± ± ±

1.02 1.00 0.51 1.00 0.52

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Table. Selected intraoperative and postoperative variables

*P < .05 compared with 0.9% normal saline (NS) treatment. Data shown as mean values ± standard error of the mean. BL, Baseline; FFP, fresh frozen plasma; Hg, hemoglobin; OB, observation; PR, post-resuscitation; PS, post-shock; VPA, valproic acid.

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Fig 4. Intracranial pressure (ICP; A) and brain tissue oxygenation (B) at selected times during the experiment. The x axis is time in minutes from initiation of TBI. Data are expressed as percentage of baseline value. FFP, Fresh frozen plasma; NS, 0.9% normal saline; VPA, valproic acid.

etiology.34,35 In contrast, some studies suggest that infusion of FFP and artificial colloids may be beneficial after TBI,36,37 and early FFP use is gaining favor as part of the massive transfusion protocols.37,38 It should be stressed that FFP is not the same as albumin. It is a complex physiologic fluid that contains a myriad of proteins, buffers, free radical scavengers, and clotting factors, and the benefits of FFP are not due simply to reversal of coagulopathy,38-40 but also to inherent cytoprotective properties of the fluid itself.16 Physiologic studies have proposed that colloids can preserve/ augment plasma oncotic pressure and thus provide better volume expansion with decreased extravasation of fluid into the brain parenchyma.41 Our findings support this concept as the FFP groups

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Fig 5. Brain injury after resuscitation with different treatments. A, Representative brain slices stained with 2,3,5triphenyltetrazolium chloride. B, Lesion size and brain swelling in different groups. Brain swelling is shown as percent increase compared with the contralateral hemisphere. FFP, Fresh frozen plasma; NS, 0.9% normal saline; VPA, valproic acid. *P < .05 compared with NS. #P < .05 FFP versus FFP+VPA.

required one third the volume for resuscitation compared with the saline group. At the molecular level, HS has been shown to cause cellular hypoacetylation, which can be reversed rapidly with histone deacetylase inhibitors, such as VPA.20,39,40 Our group has shown previously that histone deacetylase inhibitor treatment leads to decreased inflammation,18,42,43 upregulation of prosurvival pathways,42 downregulation of proapoptotic pathways,19 attenuation of ischemia–reperfusion injuries,22 and improved survival in small44 and large animal models of lethal hemorrhage/polytrauma.20 It also decreases hypoxia-induced neuronal death,23 decreases cerebral inflammation, and protects against the progression of brain injury.24 A number of important pathways are involved in creating this ‘‘prosurvival’’ phenotype.45 In the present study, the decrease in brain injury in the VPA treated animals was not owing to better resuscitation, because these animals actually had lesser MAP and slower lactate clearance. The smaller lesion size most likely reflected the enhanced ability of the vulnerable cells to survive the insult, which is similar to what has been observed in a non-TBI polytrauma model.20 This study was not designed to identify the specific underlying pathways; however, many of the mechanisms described were likely involved. Although this study was not designed to investigate all the mechanisms involved, some of these pathways may differ from the protective effects of FFP.

The high dose of VPA (300 mg/kg) was selected based on a series of previous experiments, and because the dose of VPA that can act as a histone deacetylase inhibitor is 6- to 8-fold greater than its antiseizure dose.45 Pilot studies in conjunction with our previous work,20 had shown that a rapid bolus of VPA could precipitate hypotension; therefore, we used a slower infusion rate (100 mg/kg per hour), which was found to be safe. This proof-of-concept study has some evident limitations that must be acknowledged. Although the choice of a cortical impact model may differ from common clinical injury patterns, this model offered the greatest reproducibility. The duration of HS (2 hours) was relatively long. We created this ‘‘worst case’’ scenario based on a series of pilot experiments (with increasing severity of shock) to create a large but still reversible brain lesion. Owing to the brief period of observation (6 hours), we most likely did not capture the maximum extent of the injury. This presumption is evidenced by the fact brain swelling was different between groups but ICP values were similar. With greater periods of observation, however, the differences between the groups are likely to become even more pronounced. Likewise, functional outcomes were not measured, because this was a nonsurvival study. The high dose of VPA may cause adverse neurologic effects, such as somnolence, confusion, and disorientation, as well as unresponsiveness.41,46 These effects are reversible rapidly,

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but how they may affect recovery from TBI needs to be tested further. Furthermore, the choice of resuscitation fluids may not reflect current clinical protocols, in terms of the volume as well as speed of the NS resuscitation. We used NS as a control but this study was designed primarily for a direct comparison between FFP and FFP+VPA. Additionally, the use of packed red blood cells, which is the standard for most hospital-based resuscitation, was not employed owing to several reasons. First, this study is part of a series of investigations aimed at developing novel resuscitative options for the prehospital setting where the use of packed red blood cells is challenging logistically. Second, several studies have indicated that the age of the packed red blood cells may impact on resuscitative outcomes42-44,47 and could thus potentially confound our findings. In summary, we have shown that in a large animal model of TBI+HS, early treatment with a combination of FFP and VPA decreases the size of brain lesion and associated swelling.

REFERENCES 1. Centers for Disease Control and Prevention, Injury Prevention and Control. Injury: the leading cause of death among persons aged 1-44. http://www.cdc.gov/injury/overview/ leading_cod.html. Last accessed June 4, 2013. 2. Centers for Disease Control and Prevention, National Center for Injury Prevention and Control. Web-based injury statistics query and reporting system (WISQARS). http://www. cdc.gov/traumacare/global_trauma.html. Last accessed December 2, 2012. 3. Langlois JA, Rutland-Brown W, Wald MM. The epidemiology and impact of traumatic brain injury: a brief overview. J Head Trauma Rehabil 2006;21:375-8. 4. O’Connell KM, Littleton-Kearney MT. The role of free radicals in traumatic brain injury. Biol Res Nurs. 2013 Jan 9. [Epub ahead of print]. http://www.ncbi.nlm.nih.gov/ pubmed/22345426. Last accessed June 5, 2013. 5. Schmoker JD, Zhuang J, Shackford SR. Hemorrhagic hypotension after brain injury causes an early and sustained reduction in cerebral oxygen delivery despite normalization of systemic oxygen delivery. J Trauma 1992;32:714-20. 6. McMahon CG, Yates DW, Campbell FM, Hollis S, Woodford M. Unexpected contribution of moderate traumatic brain injury to death after major trauma. J Trauma 1999;47:891-5. 7. Kelly JF, Ritenour AE, McLaughlin DF, Bagg KA, Apodaca AN, Mallak CT, et al. Injury severity and causes of death from Operation Iraqi Freedom and Operation Enduring Freedom: 2003–2004 versus 2006. J Trauma 2008;64(2 Suppl):S21-6. 8. Miller JD, Sweet RC, Narayan R, Becker DP. Early insults to the injured brain. JAMA 1978;240:439-42. 9. Wald SL, Shackford SR, Fenwick J. The effect of secondary insults on mortality and long-term disability after severe head injury in a rural region without a trauma system. J Trauma 1993;34:377-81. 10. DeWitt DS, Prough DS, Taylor CL, Whitley JM. Reduced cerebral blood flow, oxygen delivery, and electroencephalographic

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