Cecilie H. Jepsen

Assessment of coagulopathy, endothelial injury, and inflammation after traumatic brain injury and hemorrhage in a porcine model

BACKGROUND Traumatic brain injury (TBI) and hemorrhagic shock (HS) can be associated with coagulopathy and inflammation, but the mechanisms are poorly understood. We hypothesized that a combination of TBI and HS would disturb coagulation, damage the endothelium, and activate inflammatory and complement systems. METHODS A total of 33 swine were allocated to either TBI + HS (n = 27, TBI and volume-controlled 40% blood loss) or controls (n = 6, anesthesia and instrumentation). TBI + HS animals were left hypotensive (mean arterial pressure, 30–35 mm Hg) for 2 hours. Blood samples were drawn at baseline, 3 minutes and 15 minutes after injury, as well as following 2 hours of hypotension. Markers of coagulation, anticoagulation, endothelial activation/glycocalyx shedding, inflammation, complement, and sympathoadrenal function were measured. RESULTS The TBI + HS group demonstrated an immediate (3 minutes after injury) activation of coagulation (prothrombin fragment 1 + 2, 289 ng/mL vs. 232 ng/mL, p = 0.03) and complement (C5a, 2.83 ng/mL vs. 2.05 ng/mL, p = 0.05). Shedding of the endothelial glycocalyx (syndecan 1) was evident 15 minutes after injury (851.0 ng/ml vs. 715.5 ng/ml, p = 0.03) while inflammation (tumor necrosis factor &agr; [TNF-&agr;], 81.1 pg/mL vs. 50.8 pg/mL, p = 0.03) and activation of the protein C system (activated protein C, 56.7 ng/mL vs. 26.1 ng/mL, p = 0.01) were evident following the 2-hour hypotension phase. CONCLUSION The combination of TBI and shock results in an immediate activation of coagulation and complement systems with subsequent endothelial shedding, protein C activation, and inflammation.

Pharmacologic resuscitation for hemorrhagic shock combined with traumatic brain injury.

BACKGROUND We have previously demonstrated that valproic acid (VPA), a histone deacetylase inhibitor, can improve survival after hemorrhagic shock (HS), protect neurons from hypoxia-induced apoptosis, and attenuate the inflammatory response. We have also shown that administration of 6% hetastarch (Hextend [Hex]) after traumatic brain injury (TBI) decreases brain swelling, without affecting size of the lesion. This study was performed to determine whether addition of VPA to Hex would decrease the lesion size in a clinically relevant large animal model of TBI + HS. METHODS Yorkshire swine (42–50 kg) were instrumented to measure hemodynamic parameters, intracranial pressure, and brain tissue oxygenation. A custom-designed, computer-controlled cortical impact device was used to create a TBI through a 20-mm craniotomy: 15-mm cylindrical tip impactor at 4-m/s velocity, 100-millisecond dwell time, and 12-mm penetration depth. Volume-controlled hemorrhage was started (40% blood volume) concurrent with the TBI. After 2 hours of shock, animals were randomized to one of three resuscitation groups (n = 7 per group) as follows: (1) isotonic sodium chloride solution; (2) 6% hetastarch, Hex; and (3) Hex and VPA 300 mg/kg (Hex + VPA). Volumes of Hex matched the shed blood, whereas that of the isotonic sodium chloride solution was three times the volume. VPA treatment was started after an hour of shock. After 6 hours of postresuscitation monitoring, brains were 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 uninjured side). Levels of acetylated histone H3 were determined to quantify acetylation, and myeloperoxidase and interleukine-1&bgr; (IL-1&bgr;) levels were measured as markers of brain inflammation. RESULTS Combination of 40% blood loss with cortical impact and a period of shock (2 hours) and resuscitation resulted in a highly reproducible brain injury. Lesion size and brain swelling in the Hex + VPA group (1,989 [156.8] mm3, and 19% [1.6%], respectively) were significantly smaller than the isotonic sodium chloride solution group (3,335 [287.9] mm3 and 36% [2.2%], respectively). Hex alone treatment significantly decreased the swelling (27% [1.6%]) without reducing the lesion size. The number of CD11b-positive cells as well as myeloperoxidase and IL-1 levels in the brains were significantly reduced by the VPA treatment. CONCLUSION In a combined HS and TBI model, treatment with artificial colloid (Hex) improves hemodynamic parameters and reduces swelling, without affecting the actual size of the brain lesion. Addition of VPA effectively reduces both the size of brain lesion and associated swelling by attenuating the inflammatory response.

Platelet activation and dysfunction in a large-animal model of traumatic brain injury and hemorrhage

BACKGROUND: Traumatic brain injury (TBI) and hemorrhage are the leading causes of trauma‐related mortality. Both TBI and hemorrhage are associated with coagulation disturbances, including platelet dysfunction. We hypothesized that platelet dysfunction could be detected early after injury, and that this dysfunction would be associated with early activation, as measured by circulating levels of platelet activation markers. METHODS: A total of 33 swine were allocated to TBI and hypotension (n = 27, TBI and volume‐controlled 40% blood loss) or controls (n = 6, anesthesia and instrumentation only). Animals in the TBI/Hemorrhage group were left hypotensive, defined as mean arterial pressure of 35 mm Hg, for 2 hours. Blood samples were drawn at baseline and 3 minutes and 15 minutes following injury as well as following 2 hours of shock. Samples were analyzed for platelet aggregation using impedance aggregometry with agonists collagen, arachidonic acid, and adenosine diphosphate (ADP) and thromboelastography (TEG) and circulating levels of platelet activation markers transforming growth factor‐[beta] (TGF‐[beta]), CD40 ligand, and sP‐selectin. RESULTS: Platelet ADP aggregation was significantly lower in the TBI/Hemorrhage group when compared with the control group 15 minutes following injury (62.4 vs. 80.4 U, p = 0.03) as well as following 2 hours of hypotension (59.9 vs. 73.5 U, p < 0.01). The latter was associated with lower TEG measured clot strength (TEG‐MA, 74.1 vs. 79.4 mm, p = 0.05). No difference in collagen or arachidonic acid aggregation was observed. TGF‐[beta] levels were significantly higher in the TBI/Hemorrhage group following 2 hours of hypotension (1,764 vs. 1,252 pg/mL, p = 0.01). No differences in CD40 ligand or sP‐selectin levels were observed. CONCLUSION: In this combined model of TBI and hemorrhage, a significantly lower ADP‐induced platelet aggregation was detected 15 minutes following injury that was further aggravated during the 2‐hour shock period. This dysfunction was associated with an increase in platelet activation marker TGF‐[beta].

Early treatment with lyophilized plasma protects the brain in a large animal model of combined traumatic brain injury and hemorrhagic shock

BACKGROUND Combination of traumatic brain injury (TBI) and hemorrhagic shock (HS) can result in significant morbidity and mortality. We have previously shown that early administration of fresh frozen plasma (FFP) in a large animal model of TBI and HS reduces the size of the brain lesion as well as the associated edema. However, FFP is a perishable product that is not well suited for use in the austere prehospital settings. In this study, we tested whether a shelf-stable, low-volume, lyophilized plasma (LSP) product was as effective as FFP. METHODS Yorkshire swine (42–50 kg) were instrumented to measure hemodynamic parameters, intracranial pressure, and brain tissue oxygenation. A prototype, computerized, cortical impact device was used to create TBI through a 20-mm craniotomy: 15-mm cylindrical tip impactor at 4 m/s velocity, 100-millisecond dwell time, and 12-mm penetration depth. Volume-controlled hemorrhage was induced (40–45% total blood volume) concurrent with the TBI. After 2 hours of shock, animals were treated with (1) normal saline (NS, n = 5), (2) FFP (n = 5), and (3) LSP (n = 5). The volume of FFP and LSP matched the shed blood volume, whereas NS was 3 times the volume. Six hours after resuscitation, brains were sectioned and stained with TTC (2, 3, 5-Triphenyltetrazolium chloride), and lesion size (mm3) and swelling (percent change in volume compared with the contralateral, uninjured side) were measured. RESULTS This protocol resulted in a highly reproducible brain injury, with clinically relevant changes in blood pressure, cardiac output, tissue hypoperfusion, intracranial pressure, and brain tissue oxygenation. Compared with NS, treatment with LSP significantly (p < 0.05) decreased brain lesion size and swelling (51% and 54%, respectively). CONCLUSION In a clinically realistic combined TBI + HS model, early administration of plasma products decreases brain lesion size and edema. LSP is as effective as FFP, while offering many logistic advantages. LEVEL OF EVIDENCE Therapeutic study, level V.

Fresh frozen plasma resuscitation attenuates platelet dysfunction compared with normal saline in a large animal model of multisystem trauma.

BACKGROUND Platelet dysfunction following trauma has been identified as an independent predictor of mortality. We hypothesized that fresh frozen plasma (FFP) resuscitation would attenuate platelet dysfunction compared with 0.9% normal saline (NS). METHODS Twelve swine were subjected to multisystem trauma (traumatic brain injury, liver injury, rib fracture, and soft tissue injury) with hemorrhagic shock (40% of estimated blood volume). Animals were left in shock (mean arterial pressure, 30–35 mm Hg) for 2 hours followed by resuscitation with three times shed volume NS (n = 6) or one times volume FFP (n = 6) and monitored for 6 hours. Platelet function was assessed by adenosine diphosphate (ADP)–induced platelet aggregation at baseline, after 2 hours of shock following resuscitation, and 6 hours after resuscitation. Fibrinogen levels and markers of platelet activation (transforming growth factor &bgr; [TGF-&bgr;], sP-Selectin, and CD40L) as well as endothelial injury (intercellular adhesion molecule 1 [ICAM-1], vascular cell adhesion molecule 1 [VCAM-1]) were also assayed. Thromboelastography was used to measure clotting activity. RESULTS ADP-induced platelet aggregation was significantly higher in the FFP group (46.3 U vs. 25.5 U, p < 0.01) following resuscitation. This was associated with higher fibrinogen levels (202 mg/dL vs. 80 mg/dL, p < 0.01) but lower endothelial activation (VCAM-1, 1.25 ng/mL vs. 3.87 ng/mL, p = 0.05). Other markers did not differ. After 6 hours of observation, ADP-induced platelet aggregation remained higher in the FFP group (53.8 U vs. 37.0 U, p = 0.03) as was fibrinogen levels (229 mg/dL vs. 153 mg/dL, p < 0.01). Endothelial activation was lower (ICAM-1, 21.0 ng/mL vs. 24.4 ng/mL, p = 0.05), whereas TGF-&bgr; levels were higher (2,138 pg/mL vs. 1,802 pg/mL, p = 0.03) in the FFP group. Other markers did not differ. Thromboelastography revealed increased clot strength in the FFP group at both postresuscitation time points. CONCLUSION Resuscitation with FFP resulted in an immediate and sustained improvement in platelet function and clot strength compared with high-volume NS resuscitation. This was associated with an increase in fibrinogen levels and an attenuation of endothelial activation.

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

INTRODUCTION Traumatic brain injury (TBI) and hemorrhagic shock (HS) are major causes of trauma-related 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 (mm(3)) 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 mm(3) and 13 ± 1%, respectively) were less than the NS group (3,285 ± 131 mm(3) [P < .001] and 37 ± 2% [P < .001], respectively), and the FFP alone group (2,160 ± 203 mm(3) [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.

Pharmacologic modulation of cerebral metabolic derangement and excitotoxicity in a porcine model of traumatic brain injury and hemorrhagic shock

BACKGROUND Cerebral metabolic derangement and excitotoxicity play critical roles in the evolution of traumatic brain injury (TBI). We have shown previously that treatment with large doses of valproic acid (VPA) decreases the size of brain lesion. The goal of this experiment was to determine whether this effect was owing to metabolic modulation. METHODS Yorkshire swine (n = 9) underwent a protocol of computer-controlled TBI and 40% hemorrhage and were resuscitated randomly with either fresh frozen plasma equal to the volume of shed blood (FFP; n = 4) or VPA (300 mg/kg) and FFP (FFP+VPA; n = 5). Hemodynamics, brain oxygenation, and blood glucose were monitored continuously for 6 hours after resuscitation. Cerebral microdialysis was used to measure glucose, lactate, pyruvate, glutamate, and glycerol levels at baseline, 1 and 2 hours post-shock, post-resuscitation (PR), and at 2, 4, and 6 hours PR. Brain samples from the injured side were then separated into mitochondrial and cytosolic fractions, and activity of pyruvate dehydrogenase complex (PDH) was measured using a dipstick assay kit. RESULTS At baseline, there was no difference in brain lactate, pyruvate, glycerol, and glutamate concentrations between the groups. At all time points, there were no differences between the groups in brain oxygenation, cerebral perfusion pressure, or blood and brain glucose concentrations. After VPA infusion (PR time point), however, there was sustained decrease in lactate (0.91 ± 0.47 vs 2.54 ± 0.59 mmol/L; P < .01) and pyruvate (12.80 ± 4.89 vs 46.25 ± 9.22; P < .001) concentrations compared with the FFP alone group, implying superior glucose utilization for ATP production. There was also a decrease in concentrations of glutamate (6.64 ± 3.68 vs 42.25 ± 27.07 mmol/L; P = .02) and glycerol (19.20 ± 6.76 vs 69.75 ± 30.07 mmol/L; P = .01), in the FFP+VPA group, signifying lesser degree of excitotoxicity and brain damage, respectively. Brain PDH activity was greater in the mitochondrial fractions (5,984 ± 504 adjusted volume intensity [INT] × mm(2) vs 4,332 ± 1,055 INT × mm(2); P = .04) and lower in cytosolic fractions in the FFP+VPA group (1,597 ± 1,395 vs 4,026 ± 1,067 INT × mm(2); P = .03), indicating better mitochondrial membrane function and enhanced mitochondrial PDH retention. CONCLUSION VPA treatment attenuates perturbation of post-traumatic cerebral metabolism by mitigating mitochondrial dysfunction, and decreases glutamate-mediated excitotoxic damage. These properties could explain its effectiveness in decreasing lesion size and post-traumatic cerebral edema.

Fresh-frozen plasma resuscitation after traumatic brain injury and shock attenuates extracellular nucleosome levels and deoxyribonuclease 1 depletion

INTRODUCTION Traumatic brain injury and shock are among the leading causes of trauma-related mortality. We have previously shown that fresh-frozen plasma (FFP) resuscitation reduces the size of brain lesion and associated swelling compared with crystalloids. We hypothesized that this effect would be associated with an attenuation of circulating nucleosome levels, a biomarker of injury with cytotoxic potential, through reconstitution of circulating deoxyribonuclease-1 (DNAse1), an enzyme identified as critical in nucleosome clearance from the circulation. METHODS Twelve swine underwent a protocol of traumatic brain injury followed by 40% volume-controlled hemorrhage. Animals were left in shock (mean arterial pressure of 35 mmHg) for 2 hours before they were resuscitated with normal saline (NS) or FFP. Circulating levels of nucleosomes and DNAse1 were measured whereas extracellular nucleosomes were quantified on brain histology. Lesion size and brain swelling were also quantified. RESULTS Nucleosome levels were significantly greater in the NS group 6 hours after resuscitation (0.32 mU vs 0.15 mU, P = .030) whereas DNAse1 levels were substantially greater in the FFP group (9.82 ng/mL vs 4.54 ng/mL, P = .010). Circulating nucleosomes levels correlated with lesion size (rho = 0.79, P = .002) as well as brain swelling (rho = 0.89, P < .001) whereas DNAse1 levels correlated with brain swelling (rho = -0.61, P = .036) but not lesion size (rho = -0.47, P = .124). Brain staining revealed nucleosome extracellularization in both groups, but this appeared more frequent in the NS-resuscitated animals. CONCLUSION Our results show that resuscitation with FFP attenuates circulating nucleosome levels and prevents DNAse1 depletion. These factors may play a role in the neuroprotective effects observed during early resuscitation with FFP.

Effect of valproic acid and injury on lesion size and endothelial glycocalyx shedding in a rodent model of isolated traumatic brain injury.

BACKGROUND In isolated traumatic brain injury (TBI), little is known about the endothelial response and the effects of endothelial glycocalyx shedding. We have previously shown that treatment with valproic acid (VPA) improves outcomes following TBI and hemorrhagic shock. In this model, we hypothesized that severe isolated TBI would cause shedding of the endothelial glycocalyx, as measured by serum syndecan-1 (sSDC-1) levels. We further hypothesized that VPA treatment would reduce this response and reduce lesion size volume. METHODS Forty Sprague-Dawley rats were allocated to TBI + VPA (n = 8), TBI + saline vehicle control infusion (n = 8), sham + saline vehicle control infusion (n = 6), or sham + VPA (n = 8). TBI animals were subjected to severe controlled cortical impact and killed 6 hours after injury. VPA 300 mg/kg was given as an intravenous bolus 30 minutes after injury. Serum samples were analyzed for sSDC-1, and lesion size was determined on Nissl-stained cryosections. RESULTS sSDC-1 was significantly elevated in injured compared with uninjured animals at 3 hours (p = 0.0009) and 6 hours (p = 0.0007) after injury. This effect was significantly more pronounced in the animals treated with VPA (p = 0.019) 3 hours after injury, in which sSDC-1 levels were also significantly inversely correlated with lesion size (&rgr; = −0.55, p = 0.038). Lesion size was significantly smaller in TBI + VPA (40.45 mm3 ± 13.83 mm3) as compared with vehicle control (59.57 mm3 ± 16.83 mm3) (p = 0.023). CONCLUSION Severe isolated TBI caused shedding of the endothelial glycocalyx. Treatment with VPA was associated with increased glycocalyx shedding and reduced lesion size volume in injured animal.

Fresh frozen plasma resuscitation provides neuroprotection compared to normal saline in a large animal model of traumatic brain injury and polytrauma.

We have previously shown that early treatment with fresh frozen plasma (FFP) is neuroprotective in a swine model of hemorrhagic shock (HS) and traumatic brain injury (TBI). However, it remains unknown whether this strategy would be beneficial in a more clinical polytrauma model. Yorkshire swine (42-50 kg) were instrumented to measure hemodynamic parameters, brain oxygenation, and intracranial pressure (ICP) and subjected to computer-controlled TBI and multi-system trauma (rib fracture, soft-tissue damage, and liver injury) as well as combined free and controlled hemorrhage (40% blood volume). After 2 h of shock (mean arterial pressure, 30-35 mm Hg), animals were resuscitated with normal saline (NS; 3×volume) or FFP (1×volume; n=6/group). Six hours postresuscitation, brains were harvested and lesion size and swelling were evaluated. Levels of endothelial-derived vasodilator endothelial nitric oxide synthase (eNOS) and vasoconstrictor endothelin-1 (ET-1) were also measured. FFP resuscitation was associated with reduced brain lesion size (1005.8 vs. 2081.9 mm(3); p=0.01) as well as swelling (11.5% vs. 19.4%; p=0.02). Further, FFP-resuscitated animals had higher brain oxygenation as well as cerebral perfusion pressures. Levels of cerebral eNOS were higher in the FFP-treated group (852.9 vs. 816.4 ng/mL; p=0.03), but no differences in brain levels of ET-1 were observed. Early administration of FFP is neuroprotective in a complex, large animal model of polytrauma, hemorrhage, and TBI. This is associated with a favorable brain oxygenation and cerebral perfusion pressure profile as well as higher levels of endothelial-derived vasodilator eNOS, compared to normal saline resuscitation.