Valproic Acid Protects Against Acute Kidney Injury in Hemorrhage and Trauma.

INTRODUCTION: Trauma is the leading cause of death among young people. These patients have a high incidence of kidney injury, which independently increases the risk of mortality. As valproic acid (VPA) treatment has been shown to improve survival in animal models of lethal trauma, we hypothesized that it would also attenuate the degree of acute kidney injury. METHODS: We analyzed data from two separate experiments where swine were subjected to lethal insults.  Model 1: hemorrhage (50% blood volume hemorrhage followed by 72-h damage control resuscitation). Model 2: polytrauma (traumatic brain injury, 40% blood volume hemorrhage, femur fracture, rectus crush and grade V liver laceration). Animals were resuscitated with normal saline (NS) +/- VPA 150 mg/kg after a 1-h shock phase in both models (n = 5-6/group). Serum samples were analyzed for creatinine (Cr) using colorimetry on a Liasys 330 chemistry analyzer. Proteomic analysis was performed on kidney tissue sampled at the time of necropsy. RESULTS: VPA treatment significantly (P < 0.05) improved survival in both models. (Model 1: 80% vs 20%; Model 2: 83% vs. 17%). Model 1 (Hemorrhage alone): Cr increased from a baseline of 1.2 to 3.0 in NS control animals (P < 0.0001) 8 h after hemorrhage, whereas it rose only to 2.1 in VPA treated animals (P = 0.004). Model 2 (Polytrauma): Cr levels increased from baseline of 1.3 to 2.5 mg/dL (P = 0.01) in NS control animals 4 h after injury but rose to only 1.8 in VPA treated animals (P = 0.02). Proteomic analysis of kidney tissue identified metabolic pathways were most affected by VPA treatment. CONCLUSIONS: A single dose of VPA (150 mg/kg) offers significant protection against acute kidney injury in swine models of polytrauma and hemorrhagic shock.

Cl-Amidine Improves Survival and Attenuates Kidney Injury in a Rabbit Model of Endotoxic Shock.

Objective: Sepsis causes millions of deaths on a global scale annually. Activation of peptidylarginine deiminase (PAD) enzymes in sepsis causes citrullination of histones, which results in neutrophil extracellular trap formation and sepsis progression. This study evaluates pan-PAD inhibitor, Cl-amidine, in a model of lipopolysaccharide (LPS)-induced endotoxic shock in rabbits. We hypothesized that Cl-amidine would improve survival and attenuate kidney injury. Methods: In the survival model, rabbits were injected injected intravenously with 1 mg/kg of LPS, and then randomly assigned either to receive dimethyl sulfoxide (DMSO; 1 mcL/g) or Cl-amidine (10 mg/kg diluted in 1 mcL/g DMSO). They were then monitored for 14 days to evaluate survival. In the non-survival experiment, the same insult and treatment were administered, however; the animals were euthanized 12 hours after LPS injection for kidney harvest. Acute kidney injury (AKI) scoring was performed by a histopathologist who was blinded to the group assignment. Serial blood samples were also collected and compared. Results: Rabbits that received Cl-amidine had a higher survival (72%) compared with the rabbits that received DMSO (14%; p < 0.05). Cl-amidine-treated rabbits had lower (p < 0.05) histopathologic AKI scores, as well as plasma creatinine and blood urea nitrogen (BUN) levels 12 hours after insult. Conclusions: Pan-PAD inhibitor Cl-amidine improves survival and attenuates kidney injury in LPS-induced endotoxic shock in rabbits.

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 treatment rescues injured tissues after traumatic brain injury.

BACKGROUND: No agents that are specifically neuroprotective are currently approved to emergently treat patients with traumatic brain injury (TBI). The histone deacetylase inhibitor, high-dose valproic acid (VPA) has been shown to have cytoprotective potential in models of combined TBI and hemorrhagic shock, but it has not been tested in an isolated TBI model. We hypothesized that VPA, administered after isolated TBI, will penetrate the injured brain, attenuate the lesion size, and activate prosurvival pathways. METHODS: Yorkshire swine were subjected to severe TBI by cortical impact. One hour later, animals were randomized to VPA treatment (150 mg/kg delivered intravenously for 1 hour; n = 4) or control (saline vehicle; n = 4) groups. Seven hours after injury, animals were sacrificed, and brain lesion size was measured. Mass spectrometry imaging was used to visualize and quantitate brain tissue distribution of VPA. Sequential serum samples were assayed for key biomarkers and subjected to proteomic and pathway analysis. RESULTS: Brain lesion size was 50% smaller (p = 0.01) in the VPA-treated animals (3,837 ± 948 mm) compared with the controls (1,900 ± 614 mm). Endothelial regions had eightfold higher VPA concentrations than perivascular regions by mass spectrometry imaging, and it readily penetrated the injured brain tissues. Serum glial fibrillary acid protein was significantly lower in the VPA-treated compared with the control animals (p < 0.05). More than 500 proteins were differentially expressed in the brain, and pathway analysis revealed that VPA affected critical modulators of TBI response including calcium signaling pathways, mitochondria metabolism, and biosynthetic machinery. CONCLUSION: Valproic acid penetrates injured brain tissues and exerts neuroprotective and prosurvival effects that resulted in a significant reduction in brain lesion size after isolated TBI. Levels of serum biomarkers reflect these changes, which could be useful for monitoring the response of TBI patients during clinical studies.

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.