Prolonging the therapeutic window for valproic acid treatment in a swine model of traumatic brain injury and hemorrhagic shock.

BACKGROUND: It has previously been shown that administration of valproic acid (VPA) can improve outcomes if given within an hour following traumatic brain injury (TBI). This short therapeutic window (TW) limits its use in real-life situations. Based upon its pharmacokinetic data, we hypothesized that TW can be extended to 3 hours if a second dose of VPA is given 8 hours after the initial dose. METHOD: Yorkshire swine (40-45 kg; n = 10) were subjected to TBI (controlled cortical impact) and 40% blood volume hemorrhage. After 2 hours of shock, they were randomized to either (1) normal saline resuscitation (control) or (2) normal saline-VPA (150 mg/kg × two doses). First dose of VPA was started 3 hours after the TBI, with a second dose 8 hours after the first dose. Neurologic severity scores (range, 0-36) were assessed daily for 14 days, and brain lesion size was measured via magnetic resonance imaging on postinjury day 3. RESULTS: Hemodynamic and laboratory parameters of shock were similar in both groups. Valproic acid-treated animals had significantly less neurologic impairment on days 2 (16.3 ± 2.0 vs. 7.3 ± 2.8) and 3 (10.9 ± 3.6 vs. 2.8 ± 1.1) postinjury and returned to baseline levels 54% faster. Magnetic resonance imaging showed no differences in brain lesion size on day 3. Pharmacokinetic data confirmed neuroprotective levels of VPA in the circulation. CONCLUSION: This is the first study to demonstrate that VPA can be neuroprotective even when given 3 hours after TBI. This expanded TW has significant implications for the design of the clinical trial.

Prolonging the zone 1 aortic occlusion time to 4 hours using a partial resuscitative endovascular balloon in a swine model.

BACKGROUND: The clinical usage of the resuscitative endovascular balloon occlusion of the aorta (REBOA) is limited by distal ischemia resulting from complete aortic occlusion. We hypothesized that animals would physiologically tolerate the prolonged partial occlusion using the novel partially occluding REBOA (pREBOA) with survivable downstream injuries. METHODS: This study used the pREBOA-PRO catheter in a previously established swine model. Female Yorkshire swine (n = 10) underwent a volume-controlled hemorrhage (40% estimated blood). After 1 hour of shock (mean arterial pressure, 28-32 mm Hg), animals were randomized to partial occlusion for either 2 hours or 4 hours. The pREBOA was inflated in zone 1 to achieve partial occlusion defined as a distal systolic blood pressure (SBP) of 20 ± 2 mm Hg. The balloon was deflated at the end of the occlusion period, and animals were resuscitated for 2 hours. Tissues were examined for gross and histologic injury. The primary endpoint was histologic organ injury, and secondary end points were hemodynamic variables and degree of distal organ ischemia. RESULTS: All animals survived to the endpoint. Both groups had similar proximal and distal SBP at baseline, with a divergence of pressures ranging from 55 mm Hg to 90 mm Hg on inflation. The lactate levels increased throughout the occlusion and decreased approximately 40% during the observation period. More animals required norepinephrine and fluid in the 4-hour group compared with the 2-hour group. There was no gross small bowel ischemia noted in the 2-hour animals. The 4-hour group had surgically resectable patchy short segment ischemia. Neither group showed nonsurvivable organ ischemia on pathology or laboratory values. CONCLUSION: This is the first study showing that the zone 1 aorta can be occluded for over 4 hours using a new pREBOA device without need for balloon titration. In conclusion, simple changes in balloon design offer reliable partial aortic occlusion, with potentially survivable and surgically manageable downstream injuries.

Recombinant human MG53 protein attenuates brain lesion size in a large animal model of traumatic brain injury.

BACKGROUND: MG53, a member of the tripartite motif (TRIM) protein family, plays an essential role in cell membrane repair and promotes cell survival. Recent studies show that systemic delivery of recombinant human MG53 (rhMG53) protein markedly attenuates tissue injury/inflammation, and facilitates healing. This study was performed to test whether intravenous administration of rhMG53 protein would decrease the lesion size in a clinically relevant large animal model of traumatic brain injury (TBI). METHOD: Yorkshire swine (40-45 kg; n = 5/group) were subjected to controlled cortical impact TBI and randomized to either: (1) rhMG53 protein (2 mg/kg, intravenous) or (2) normal saline control. Hemodynamics, intracranial pressure, and brain oxygenation were monitored for 7 hours. Brains were then harvested and sectioned into 5-mm slices and stained with 2,3,5-triphenyltetrazolium chloride to quantify the lesion size. Blood-brain barrier permeability of MG53 in the brain was determined by Western blot and immunohistochemistry. Bcl-2 and phospho-GSK ß levels were measured as makers of prosurvival pathway activation. RESULTS: Hemodynamic parameters were similar in both groups, but the lesion size in the rhMG53-treated group (2,517 ± 525.4 mm 3 ) was significantly ( p < 0.05) smaller than the control group (3,646 ± 740.1 mm 3 ). In the treated animals, rhMG53 was detected in the regions surrounding the TBI, but it was absent in the saline-treated control animals. Bcl-2 and phospho-GSK ß levels in the brains were upregulated in the rhMG53-treated animals. CONCLUSION: Intravenously administered rhMG53 localizes to the injured areas of the brain, with the treated animals demonstrating a significant attenuation in the brain lesion size following TBI.

A single dose of valproic acid improves neurologic recovery and decreases brain lesion size in swine subjected to an isolated traumatic brain injury.

BACKGROUND: We lack specific treatments for traumatic brain injury (TBI), which remains the leading cause of trauma-related morbidity and mortality. Treatment with valproic acid (VPA) improves outcomes in models of severe TBI with concurrent hemorrhage. However, it is unknown if VPA will have similar benefits after isolated nonlethal TBI, which is the more common clinical scenario. The goal of this study was to evaluate the effect of VPA treatment in a preclinical isolated TBI swine model on neurologic outcomes and brain lesion size and to perform detailed pharmacokinetic analyses for a future clinical trial. METHODS: Yorkshire swine (n = 10; 5/cohort) were subjected to TBI (8-mm controlled cortical impact). An hour later, we randomized them to receive VPA (150 mg/kg) or saline placebo (control). Neuroseverity scores were assessed daily (0 [normal] to 36 [comatose]), brain lesion size was measured on postinjury 3, and serial blood samples were collected for pharmacokinetic studies. RESULTS: Physiologic parameters and laboratory values were similar in both groups. Valproic acid-treated animals demonstrated significantly better neuroseverity scores on postinjury 1 (control, 9.2 ± 4.4; VPA, 0 ± 0; p = 0.001). Valproic acid-treated animals had significantly smaller brain lesion sizes (mean volume in microliter: control, 3,130 ± 2,166; VPA, 764 ± 208; p = 0.02). Pharmacokinetic data confirmed adequate plasma and tissue levels of VPA. CONCLUSION: In this clinically relevant model of isolated TBI, a single dose of VPA attenuates neurological impairment and decreases brain lesion size.

Development of a large animal model of lethal polytrauma and intra-abdominal sepsis with bacteremia.

BACKGROUND: Trauma and sepsis are individually two of the leading causes of death worldwide. When combined, the mortality is greater than 50%. Thus, it is imperative to have a reproducible and reliable animal model to study the effects of polytrauma and sepsis and test novel treatment options. Porcine models are more translatable to humans than rodent models due to the similarities in anatomy and physiological response. We embarked on a study to develop a reproducible model of lethal polytrauma and intra-abdominal sepsis, which was lethal, though potentially salvageable with treatment. METHODS: Our laboratory has a well-established porcine model that was used as the foundation. Animals were subjected to a rectus crush injury, long bone fracture, liver and spleen laceration, traumatic brain injury and hemorrhage that was used as a foundation. We tested various colon injuries to create intra-abdominal sepsis. All animals underwent injuries followed by a period of shock, then subsequent resuscitation. RESULTS: All animals had blood culture-proven sepsis. Attempts at long-term survival of animals after injury were ceased because of poor appetite and energy. We shifted to an 8-hour endpoint. The polytrauma injury pattern remained constant and the colon injury pattern changed with the intention of creating a model that was ultimately lethal but potentially salvageable with a therapeutic drug. An uncontrolled cecal injury (n=4) group resulted in very early deaths. A controlled cecal injury (CCI; n=4) group had prolonged time prior to mortality with one surviving to the endpoint. The sigmoid injury (n=5) produced a similar survival curve to CCI but no animals surviving to the endpoint. CONCLUSION: We have described a porcine model of polytrauma and sepsis that is reproducible and may be used to investigate novel treatments for trauma and sepsis. LEVEL OF EVIDENCE: Not applicable. Animal study.

A novel partial resuscitative endovascular balloon aortic occlusion device that can be deployed in zone 1 for more than 2 hours with minimal provider titration.

BACKGROUND: Hemorrhage is a leading cause of mortality in trauma. Resuscitative endovascular balloon occlusion of the aorta (REBOA) can control hemorrhage, but distal ischemia, subsequent reperfusion injury, and the need for frequent balloon titration remain problems. Improved device design can allow for partial REBOA (pREBOA) that may provide hemorrhage control while also perfusing distally without need for significant provider titration. METHODS: Female Yorkshire swine (N = 10) were subjected to 40% hemorrhagic shock for 1 hour (mean arterial pressure [MAP], 28-32 mm Hg). Animals were then randomized to either complete aortic occlusion (ER-REBOA) or partial occlusion (novel pREBOA-PRO) without frequent provider titration or distal MAP targets. Detection of a trace distal waveform determined partial occlusion in the pREBOA-PRO arm. After 2 hours of zone 1 occlusion, the hemorrhaged whole blood was returned. After 50% autotransfusion, the balloon was deflated over a 10-minute period. Following transfusion, the animals were survived for 2 hours while receiving resuscitation based on objective targets: lactated Ringer's fluid boluses (goal central venous pressure, ≥ 6 mm Hg), a norepinephrine infusion (goal MAP, 55-60 mm Hg), and acid-base correction (goal pH, >7.2). Hemodynamic variables, arterial lactate, lactate dehydrogenase, aspartate aminotransferase, and creatinine levels were measured. RESULTS: All animals survived throughout the experiment, with similar increase in proximal MAPs in both groups. Animals that underwent partial occlusion had slightly higher distal MAPs. At the end of the experiment, the partial occlusion group had lower end levels of serum lactate (p = 0.006), lactate dehydrogenase (p = 0.0004) and aspartate aminotransferase (p = 0.004). Animals that underwent partial occlusion required less norepinephrine (p = 0.002), less bicarbonate administration (p = 0.006), and less fluid resuscitation (p = 0.042). CONCLUSION: Improved design for pREBOA can decrease the degree of distal ischemia and reperfusion injury compared with complete aortic occlusion, while providing a similar increase in proximal MAPs. This can allow pREBOA zone-1 deployment for longer periods without the need for significant balloon titration.

Administration of valproic acid in clinically approved dose improves neurologic recovery and decreases brain lesion size in swine subjected to hemorrhagic shock and traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) and hemorrhage remain the leading causes of death after trauma. We have previously shown that a dose of valproic acid (VPA) at (150 mg/kg) can decrease brain lesion size and hasten neurologic recovery. The current Food and Drug Administration-approved dose of VPA is 60 mg/kg. We evaluate neurologic outcomes and brain lesion size of a single dose of VPA at a level currently within Food and Drug Administration-approved dose in swine subjected to TBI and hemorrhagic shock. METHODS: Swine (n = 5/group) were subjected to TBI and 40% blood volume hemorrhage. Animals remained in shock for 2 hours before randomization to normal saline (NS) resuscitation alone (control), NS-VPA 150 mg/kg (VPA 150), or NS-VPA 50 mg/kg (VPA 50). Neurologic severity scores (range, 0-32) were assessed daily for 14 days, and brain lesion size was measured via magnetic resonance imaging on postinjury day (PID) 3. RESULTS: Shock severity and laboratory values were similar in all groups. Valproic acid-treated animals demonstrated significantly less neurologic impairment on PID 1 and returned to baseline faster (PID 1 mean neurologic severity score, control = 22 ± 3 vs. VPA 150 mg/kg = 8 ± 7 or VPA 50 mg/kg = 6 ± 6; p = 0.02 and 0.003). Valproic acid-treated animals had significantly smaller brain lesion sizes (mean volume in mm3, control = 1,268.0 ± 241.2 vs. VPA 150 mg/kg = 620.4 ± 328.0 or VPA 50 mg/kg = 438.6 ± 234.8; p = 0.007 and 0.001). CONCLUSION: In swine subjected to TBI and hemorrhagic shock, VPA treatment, in a dose that is approved for clinical use, decreases brain lesion size and reduces neurologic impairment compared with resuscitation alone.

Valproic acid decreases resuscitation requirements after hemorrhage in a prolonged damage-control resuscitation model.

BACKGROUND: Hemorrhage is the leading cause of preventable death in trauma. Future military conflicts are likely to be in austere environments, where prolonged damage-control resuscitation (p-DCR) may be required for 72 hours before evacuation. There is a need to demonstrate that p-DCR is feasible and to optimize its logistics. Dried plasma (DP) is a practical alternative to conventional blood products in austere settings, and valproic acid (VPA) improves survival in preclinical models of trauma and hemorrhage. We performed the current experiment to study the synergistic effects of VPA and DP and hypothesized that VPA treatment would decrease the fluid resuscitation requirements in p-DCR. METHODS: Female swine were subjected to 50% hemorrhage (associated with 20% survival using non-plasma-based p-DCR) and left unresuscitated for 1 hour to simulate medic response time. They were then randomized to receive VPA (150 mg/kg + DP 250 mL; DP-VPA group; n = 5) or DP alone (DP group; n = 6). All animals were resuscitated to a systolic blood pressure of 80 mm Hg with lactated Ringer according to the Tactical Combat Casualty Care Guidelines for 72 hours, after which packed red blood cells were transfused to simulate evacuation to higher levels of care. RESULTS: The DP-VPA group needed significantly (p = 0.002) less volume of lactated Ringer to reach and maintain the target systolic blood pressure. This would translate to a 4.3 L volume sparing effect for a 70-kg person. CONCLUSION: Addition of a single dose of VPA significantly decreases the volume of resuscitation required in a p-DCR model.

Early single-dose exosome treatment improves neurologic outcomes in a 7-day swine model of traumatic brain injury and hemorrhagic shock.

BACKGROUND: Early single-dose treatment with human mesenchymal stem cell-derived exosomes promotes neuroprotection and promotes blood-brain barrier integrity in models of traumatic brain injury (TBI) and hemorrhagic shock (HS) in swine. The impact of an early single dose of exosomes on late survival (7 days), however, remains unknown. We sought to evaluate the impact of early single-dose exosome treatment on neurologic outcomes, brain lesion size, inflammatory cytokines, apoptotic markers, and mediators of neural plasticity in a 7-day survival model. METHODS: Yorkshire swine were subjected to a severe TBI (8-mm cortical impact) and HS (40% estimated total blood volume). After 1 hour of shock, animals were randomized (n = 4/cohort) to receive either lactated Ringer's (5 mL) or lactated Ringer's with exosomes (1 × 10 exosome particles). After an additional hour of shock, animals were resuscitated with normal saline. Daily neurologic severity scores were compared. At 7 days following injury, lesion size, inflammatory markers, and mediators of inflammation (NF-κB), apoptosis (BAX), and neural plasticity (brain-derived neurotrophic factor) in brain tissue were compared between groups. RESULTS: Exosome-treated animals had significantly lower neurologic severity scores (first 4 days; p < 0.05) and faster neurologic recovery. At 7 days, exosome-treated animals had significantly smaller (p < 0.05) brain lesion sizes. Exosome-treated animals also had significantly lower levels of inflammatory markers (interleukin [IL]-1, IL-6, IL-8, and IL-18) and higher granulocyte-macrophage colony-stimulating factor levels compared with the control animals, indicating specific impacts on various cytokines. The BAX and NF-κB levels were significantly lower (p < 0.05) in exosome-treated animals, while brain-derived neurotrophic factor levels were significantly higher (p < 0.05) in the exosome-treated animals. CONCLUSION: In a large animal model of TBI and HS, early single-dose exosome treatment attenuates neurologic injury, decreases brain lesion size, inhibits inflammation and apoptosis, and promotes neural plasticity over a 7-day period.

Histone deacetylase 6 inhibition improves survival in a swine model of lethal hemorrhage, polytrauma, and bacteremia.

BACKGROUND: Trauma is the leading cause of death for young Americans. Nonspecific histone deacetylase inhibitors, such as valproic acid, have been shown to improve survival in preclinical models of lethal trauma, hemorrhage, and sepsis. The doses needed to achieve a survival benefit are higher than Food and Drug Administration-approved doses, and the nonspecificity raises concerns about unintended adverse effects. The isoform-specific histone deacetylase 6 inhibitor, ACY-1083, has been found to be as efficacious as valproic acid in a rodent model of hemorrhagic shock. We hypothesized that ACY-1083 treatment would improve survival in a swine model of lethal hemorrhage, polytrauma, and bacteremia. METHODS: Swine were subjected to 45% blood volume hemorrhage, brain injury, femur fracture, rectus crush, splenic and liver lacerations, and colon injury. After 1 hour of shock (mean arterial pressure, 30-35 mm Hg), animals were randomized to normal saline resuscitation (control) or normal saline plus ACY-1083 30 mg/kg treatment (n = 5/group). After 3 hours (simulating delayed evacuation), packed red blood cells and antibiotics were administered, the colon injury was repaired, and the abdomen was closed. Animals were then monitored for another 4 hours. Survival was assessed using Kaplan-Meier and log-rank test. RESULTS: This combination of injuries was lethal. All animals became bacteremic, in addition to the severe hemorrhagic shock. Survival in the control group was 0%, and ACY-1083 treatment increased survival to 80% (p = 0.019). There was no difference in the brain lesion size between the groups. CONCLUSION: A single dose of ACY-1083 markedly improves survival in an otherwise lethal model of polytrauma, hemorrhagic shock, and bacteremia.

Early single-dose treatment with exosomes provides neuroprotection and improves blood-brain barrier integrity in swine model of traumatic brain injury and hemorrhagic shock.

BACKGROUND: Administration of human mesenchymal stem cell (MSC)-derived exosomes can enhance neurorestoration in models of traumatic brain injury (TBI) and hemorrhagic shock (HS). The impact of early treatment with MSC-derived exosomes on brain injury in a large animal model remains unknown. We sought to evaluate the impact of early single-dose exosome treatment on brain swelling and lesion size, blood-based cerebral biomarkers, and blood-brain barrier (BBB) integrity. METHODS: Female Yorkshire swine were subjected to a severe TBI (12-mm cortical impact) and HS (40% estimated total blood volume). One hour into shock, animals were randomized (n = 5/cohort) to receive either lactated Ringer's (LR; 5 mL) or LR + exosomes (1 × 10 exosome particles in 5 mL LR). Animals then underwent additional shock (1 hour) followed by normal saline resuscitation. After 6 hours of observation, brain swelling (% increase compared with the uninjured side) and lesion size (mm) were assessed. Cerebral hemodynamics and blood-based biomarkers of brain injury were compared. Immunofluorescence and RNA sequencing with differential gene expression and pathway analysis were used to assess the integrity of the perilesion BBB. RESULTS: Exosome-treated animals had significantly less (p < 0.05) brain swelling and smaller lesion size. They also had significantly decreased (p < 0.05) intracranial pressures and increased cerebral perfusion pressures. Exosome-treated animals had significantly decreased (p < 0.05) albumin extravasation and significantly higher (p < 0.05) laminin, claudin-5, and zonula occludens 1 levels. Differential gene expression and pathway analysis confirmed these findings. Serum glial fibrillary acidic protein levels were also significantly lower (p < 0.05) in the exosome-treated cohort at the end of the experiment. CONCLUSION: In a large animal model of TBI and HS, early treatment with a single dose of MSC-derived exosomes significantly attenuates brain swelling and lesion size, decreases levels of blood-based cerebral biomarkers, and improves BBB integrity.

Dose optimization of valproic acid in a lethal model of traumatic brain injury, hemorrhage, and multiple trauma in swine.

BACKGROUND: Trauma is a leading cause of death, and traumatic brain injury is one of the hallmark injuries of current military conflicts. Valproic acid (VPA) administration in high doses (300-400 mg/kg) improves survival in lethal trauma models, but effectiveness of lower doses on survival is unknown. This information is essential for properly designing the upcoming clinical trials. We, therefore, performed the current study to determine the lowest dose at which VPA administration improves survival in a model of lethal injuries. METHODS: Swine were subjected to traumatic brain injury (10-mm cortical impact), 40% blood volume hemorrhage, and multiple trauma (femur fracture, rectus crush, and Grade V liver laceration). After 1 hour of shock, animals were randomized (n = 6/group) to four groups: normal saline (NS) resuscitation; or NS with VPA doses of 150 mg/kg (VPA 150) or 100 mg/kg (VPA 100) administered over 3 hours or 100 mg/kg over 2 hours (VPA 100 over 2 hours). Three hours after shock, packed red blood cells were given, and animals were monitored for another 4 hours. Survival was assessed using Kaplan-Meier and log-rank test. RESULTS: Without resuscitation, all of the injured animals died within 5 hours. Similar survival rates were observed in the NS (17%) and VPA 100 (0%) resuscitation groups. Survival rates in the 100-mg/kg VPA groups were significantly (p < 0.05) better when it was given over 2 hours (67%) compared to 3 hours (0%). 83% of the animals in the VPA 150 group survived, which was significantly higher than the NS and VPA 100 over 3 hours groups (p < 0.05). CONCLUSION: A single dose of VPA (150 mg/kg) significantly improves survival in an otherwise lethal model of multiple injuries. This is a much lower dose than previously shown to have a survival benefit and matches the dose that is tolerated by healthy human subjects with minimal adverse effects. LEVEL OF EVIDENCE: Therapeutic, level V.

Valproic acid improves survival and decreases resuscitation requirements in a swine model of prolonged damage control resuscitation.

BACKGROUND: Although damage control resuscitation (DCR) is routinely performed for short durations, prolonged DCR may be required in military conflicts as a component of prolonged field care. Valproic acid (VPA) has been shown to have beneficial properties in lethal hemorrhage/trauma models. We sought to investigate whether the addition of a single dose of VPA to a 72-hour prolonged DCR protocol would improve clinical outcomes. METHODS: Fifteen Yorkshire swine (40-45 kg) were subjected to lethal (50% estimated total blood volume) hemorrhagic shock (HS) and randomized to three groups: (1) HS, (2) HS-DCR, (3) HS-DCR-VPA (150 mg/kg over 3 hours) (n = 5/cohort). In groups assigned to receive DCR, Tactical Combat Casualty Care guidelines were applied (1 hour into the shock period), targeting a systolic blood pressure of 80 mm Hg. At 72 hours, surviving animals were given transfusion of packed red blood cells, simulating evacuation to higher echelons of care. Survival rates, physiologic parameters, resuscitative fluid requirements, and laboratory profiles were used to compare the clinical outcomes. RESULTS: This model was 100% lethal in the untreated animals. DCR improved survival to 20%, although this was not statistically significant. The addition of VPA to DCR significantly improved survival to 80% (p < 0.01). The VPA-treated animals also had significantly (p < 0.05) higher systolic blood pressures, lower fluid resuscitation requirements, higher hemoglobin levels, and lower creatinine and potassium levels. CONCLUSION: VPA administration improves survival, decreases resuscitation requirements, and improves hemodynamic and laboratory parameters when added to prolonged DCR in a lethal hemorrhage model.

Traumatic brain injury may worsen clinical outcomes after prolonged partial resuscitative endovascular balloon occlusion of the aorta in severe hemorrhagic shock model.

BACKGROUND: The use of partial resuscitative endovascular balloon occlusion of the aorta (pREBOA) in combined hemorrhagic shock (HS) and traumatic brain injury (TBI) has not been well studied. We hypothesized that the use of pREBOA in the setting of TBI would be associated with worse clinical outcomes. METHODS: Female Yorkshire swine were randomized to the following groups: HS-TBI, HS-TBI-pREBOA, and HS-pREBOA (n = 5/cohort). Animals in the HS-TBI group were left in shock for a total of 2 hours, whereas animals assigned to pREBOA groups were treated with supraceliac pREBOA deployment (60 minutes) 1 hour into the shock period. All animals were then resuscitated, and physiologic parameters were monitored for 6 hours. Further fluid resuscitation and vasopressors were administered as needed. At the end of the observation period, brain hemispheric swelling (%) and lesion size (mm) were assessed. RESULTS: Mortality was highest in the HS-TBI-pREBOA group (40% [2/5] vs. 0% [0/5] in the other groups, p = 0.1). Severity of shock was greatest in the HS-TBI-pREBOA group, as defined by peak lactate levels and pH nadir (p < 0.05). Fluid resuscitation and norepinephrine requirements were significantly higher in the HS-TBI-pREBOA group (p < 0.05). No significant differences were noted in brain hemispheric swelling and lesion size between the groups. CONCLUSION: Prolonged application of pREBOA in the setting of TBI does not contribute to early worsening of brain lesion size and edema. However, the addition of TBI to HS-pREBOA may worsen the severity of shock. Providers should be aware of the potential physiologic sequelae induced by TBI.

Mesenchymal Stem Cell-Derived Exosomes Provide Neuroprotection and Improve Long-Term Neurologic Outcomes in a Swine Model of Traumatic Brain Injury and Hemorrhagic Shock.

Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) remains a leading cause of preventable death worldwide. Mesenchymal stem cell-derived exosomes have demonstrated promise in small animal models of neurologic injury. To investigate the effects of exosome treatment in a clinically realistic large animal model, Yorkshire swine underwent TBI and HS. Animals were maintained in shock for 2 h before resuscitation with normal saline (NS). Animals were then resuscitated either with NS (3 × volume of shed blood) or with the same volume of NS with delayed exosome administration (1 × 1013 particles/4 mL) (n = 5/cohort). Exosomes were administered 9 h post-injury, and on post-injury days (PID) 1, 5, 9, and 13. Neurologic severity scores (NSS) were assessed for 30 days, and neurocognitive functions were objectively measured. Exosome-treated animals had significantly lower NSS (p < 0.05) during the first five days of recovery. Exosome-treated animals also had a significantly shorter time to complete neurologic recovery (NSS = 0) compared with animals given NS alone (days to recovery: NS = 16.8 ± 10.6; NS + exosomes = 5.6 ± 2.8; p = 0.03). Animals treated with exosomes initiated neurocognitive testing earlier (days to initiation: NS = 9.6 ± 0.5 vs. NS + exosomes = 4.2 ± 0.8; p = 0.008); however, no difference was seen in time to mastery of tasks. In conclusion, treatment with exosomes attenuates the severity of neurologic injury and allows for faster neurologic recovery in a clinically realistic large animal model of TBI and HS.

Isoform 6-selective histone deacetylase inhibition reduces lesion size and brain swelling following traumatic brain injury and hemorrhagic shock.

BACKGROUND: Nonselective histone deacetylase (pan-HDAC) inhibitors, such as valproic acid (VPA), have demonstrated neuroprotective properties in trauma models. However, isoform-specific HDAC inhibitors may provide opportunity for more effective drug administration with fewer adverse effects. We investigated HDAC6 inhibition with ACY-1083 in an in vitro and an in vivo large animal model of injury. METHODS: Mouse hippocampal cells were subjected to oxygen-glucose deprivation (0% O2, glucose-free and serum-free medium, 18 hours) and reoxygenation (21% O2, normal culture media, 4 hours) with/without VPA (4 mmol/L) or ACY-1083 (30 nmol/L, 300 nmol/L). Cell viability was measured by methylthiazolyl tetrazolium assay. Expression of hypoxia-inducible factor-1α, heat shock protein 70, and effectors in the phosphoinositide-3 kinase/mammalian target of rapamycin pathway were measured by Western blot analysis. Additionally, swine were subjected to combined traumatic brain injury and hemorrhagic shock and randomized to three treatment groups (n = 5/group): (i) normal saline (NS; 3× hemorrhage volume); (ii) NS + VPA (NS; 3× hemorrhage volume, VPA; 150 mg/kg), and (iii) NS + ACY-1083 (NS; 3× hemorrhage volume, ACY-1083; 30 mg/kg). After 6 hours, brain tissue was harvested to assess lesion size and brain swelling. RESULTS: Significant improvement in cell viability was seen with both HDAC inhibitors in the in vitro study. ACY-1083 suppressed hypoxia-inducible factor-1α expression and up-regulated phosphorylated mammalian target of rapamycin and heat shock protein 70 in a dose-dependent manner. Lesion size and brain swelling in animals treated with pharmacologic agents (VPA and ACY-1083) were both smaller than in the NS group. No differences were observed between the VPA and ACY-1083 treatment groups. CONCLUSIONS: In conclusion, selective inhibition of HDAC6 is as neuroprotective as nonselective HDAC inhibition in large animal models of traumatic brain injury and hemorrhagic shock.

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine.

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the forefront of trauma care in recent years. Since complete aortic occlusion presents serious concerns, the concept of partial aortic occlusion has gained a growing attention. Here, we present a large animal model of hemorrhagic shock to investigate the effects of a novel partial aortic balloon occlusion catheter and compare it with a catheter that works on the principles of complete aortic occlusion. Swine are anesthetized and instrumented in order to conduct controlled fixed-volume hemorrhage, and hemodynamic and physiological parameters are monitored. Following hemorrhage, aortic balloon occlusion catheters are inserted and inflated in the supraceliac aorta for 60 min, during which the animals receive whole-blood resuscitation as 20% of the total blood volume (TBV). Following balloon deflation, the animals are monitored in a critical care setting for 4 h, during which they receive fluid resuscitation and vasopressors as needed. The partial aortic balloon occlusion demonstrated improved distal mean arterial pressures (MAPs) during the balloon inflation, decreased markers of ischemia, and decreased fluid resuscitation and vasopressor use. As swine physiology and homeostatic responses following hemorrhage have been well-documented and are like those in humans, a swine hemorrhagic shock model can be used to test various treatment strategies. In addition to treating hemorrhage, aortic balloon occlusion catheters have become popular for their role in cardiac arrest, cardiac and vascular surgery, and other high-risk elective surgical procedures.

Valproic acid induces prosurvival transcriptomic changes in swine subjected to traumatic injury and hemorrhagic shock.

BACKGROUND: Valproic acid (VPA) is a histone deacetylase inhibitor that improves outcomes in large animal models of trauma. However, its protective mechanism of action is not completely understood. We sought to characterize the genetic changes induced by VPA treatment following traumatic injuries. METHODS: Six female Yorkshire swine were subjected to traumatic brain injury (controlled cortical impact), polytrauma (liver and splenic laceration, rib fracture, rectus crush), and hemorrhagic shock (HS, 40% total blood volume). Following 2 hours of HS, animals were randomized to resuscitation with normal saline (NS) or NS + 150 mg/kg of intravenous VPA (n = 3/cohort, 18 samples total). Blood samples were collected for isolation of peripheral blood mononuclear cells at three distinct time points: baseline, 6 hours following injuries, and on postinjury day 1. RNA was extracted from peripheral blood mononuclear cells and sequenced. Differential expression analysis (false discovery rate < 0.001 and p value <0.001) and gene set enrichment (Panther Gene Ontology and Ingenuity Pathway Analysis) was used to compare VPA to non-VPA-treated animals. RESULTS: A total of 628 differentially expressed RNA transcripts were identified, 412 of which were used for analysis. There was no difference between treatment groups at baseline. The VPA-induced genetic changes were similar at 6 hours and on postinjury day 1. Upregulated genes were associated with gene expression (p 2.13E-34), cellular development (1.19E-33), cellular growth and proliferation (1.25E-30), and glucocorticoid receptor signaling (8.6E-21). Downregulated genes were associated with cell cycle checkpoint regulation (3.64E-22), apoptosis signaling (6.54E-21), acute phase response signaling (5.84E-23), and the inflammasome pathway (1.7E-19). CONCLUSION: In injured swine, VPA increases the expression of genes associated with cell survival, proliferation, and differentiation and decreases those associated with cell death and inflammation. These genetic changes could explain the superior clinical outcomes in VPA-treated animals, including smaller brain lesion size and improved neurologic recovery.

Valproic acid decreases brain lesion size and improves neurologic recovery in swine subjected to traumatic brain injury, hemorrhagic shock, and polytrauma.

BACKGROUND: We have previously shown that treatment with valproic acid (VPA) decreases brain lesion size in swine models of traumatic brain injury (TBI) and controlled hemorrhage. To translate this treatment into clinical practice, validation of drug efficacy and evaluation of pharmacologic properties in clinically realistic models of injury are necessary. In this study, we evaluate neurologic outcomes and perform pharmacokinetic analysis of a single dose of VPA in swine subjected to TBI, hemorrhagic shock, and visceral hemorrhage. METHODS: Yorkshire swine (n = 5/cohort) were subjected to TBI, hemorrhagic shock, and polytrauma (liver and spleen injury, rib fracture, and rectus abdominis crush). Animals remained in hypovolemic shock for 2 hours before resuscitation with isotonic sodium chloride solution (ISCS; volume = 3× hemorrhage) or ISCS + VPA (150 mg/kg). Neurologic severity scores were assessed daily for 30 days, and brain lesion size was measured via magnetic resonance imaging on postinjury days (PID) 3 and 10. Serum samples were collected for pharmacokinetic analysis. RESULTS: Shock severity and response to resuscitation were similar in both groups. Valproic acid-treated animals demonstrated significantly less neurologic impairment between PID 1 to 5 and smaller brain lesions on PID 3 (mean lesion size ± SEM, mm: ISCS = 4,956 ± 1,511 versus ISCS + VPA = 828 ± 279; p = 0.047). No significant difference in lesion size was identified between groups at PID 10 and all animals recovered to baseline neurologic function during the 30-day observation period. Animals treated with VPA had faster neurocognitive recovery (days to initiation of testing, mean ± SD: ISCS = 6.2 ± 1.6 vs ISCS + VPA = 3.6 ± 1.5; p = 0.002; days to task mastery: ISCS = 7.0 ± 1.0 vs ISCS + VPA = 4.8 ± 0.5; p = 0.03). The mean ± SD maximum VPA concentrations, area under the curve, and half-life were 145 ± 38.2 mg/L, 616 ± 150 hour·mg/L, and 1.70 ± 0.12 hours. CONCLUSIONS: In swine subjected to TBI, hemorrhagic shock, and polytrauma, VPA treatment is safe, decreases brain lesion size, and reduces neurologic injury compared to resuscitation with ISCS alone. These benefits are achieved at clinically translatable serum concentrations of VPA. LEVEL OF EVIDENCE: Therapeutic (preclinical study).

Addition of low-dose valproic acid to saline resuscitation provides neuroprotection and improves long-term outcomes in a large animal model of combined traumatic brain injury and hemorrhagic shock.

BACKGROUND: Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) is highly lethal. In a nonsurvival model of TBI + HS, addition of high-dose valproic acid (VPA) (300 mg/kg) to hetastarch reduced brain lesion size and associated swelling 6 hours after injury; whether this would have translated into better neurologic outcomes remains unknown. It is also unclear whether lower doses of VPA would be neuroprotective. We hypothesized that addition of low-dose VPA to normal saline (NS) resuscitation would result in improved long-term neurologic recovery and decreased brain lesion size. METHODS: TBI was created in anesthetized swine (40-43 kg) by controlled cortical impact, and volume-controlled hemorrhage (40% volume) was induced concurrently. After 2 hours of shock, animals were randomized (n = 5 per group) to NS (3× shed blood) or NS + VPA (150 mg/kg). Six hours after resuscitation, packed red blood cells were transfused, and animals were recovered. Peripheral blood mononuclear cells were analyzed for acetylated histone-H3 at lysine-9. A Neurological Severity Score (NSS) was assessed daily for 30 days. Brain magnetic resonance imaging was performed on Days 3 and 10. Cognitive performance was assessed by training animals to retrieve food from color-coded boxes. RESULTS: There was a significant increase in histone acetylation in the NS + VPA-treated animals compared with NS treatment. The NS + VPA group demonstrated significantly decreased neurologic impairment and faster speed of recovery as well as smaller brain lesion size compared with the NS group. Although the final cognitive function scores were similar between the groups, the VPA-treated animals reached the goal significantly faster than the NS controls. CONCLUSION: In this long-term survival model of TBI + HS, addition of low-dose VPA to saline resuscitation resulted in attenuated neurologic impairment, faster neurologic recovery, smaller brain lesion size, and a quicker normalization of cognitive functions.