LR Resuscitation Research Articles

Normal saline versus lactated Ringer’s solution for acute pancreatitis resuscitation, an open-label multicenter randomized controlled trial: the WATERLAND trial study protocol

BackgroundSome evidence suggests that fluid resuscitation with lactated Ringer’s solution (LR) may have an anti-inflammatory effect on acute pancreatitis (AP) when compared to normal saline (NS) and may be associated with a decrease in severity, but existing single-center randomized controlled trials showed conflicting results. The WATERLAND trial aims to investigate the efficacy and safety of fluid resuscitation using LR compared to NS in patients with AP.MethodsThe WATERLAND trial is an international multicenter, open-label, parallel-group, randomized, controlled, superiority trial. Patients will be randomly assigned in a 1:1 ratio to receive LR versus NS-based fluid resuscitation for at least 48 h. The primary outcome will be moderately severe or severe AP, according to the revision of the Atlanta classification. The secondary objectives of the WATERLAND trial are to determine the effect of LR versus NS fluid resuscitation on several efficacy and safety outcomes in patients with AP.A total sample of 720 patients, 360 in the LR group and 360 in the NS group, will achieve 90% power to detect a difference between the group proportions of 10%, assuming that the frequency of moderately severe or severe AP in the LR group will be 17%. A loss to follow-up of 10% of patients is expected, so the total sample size will be 396 patients in each treatment arm (792 patients overall). The test statistic used is the two-sided Z test with pooled variance set at a 0.05 significance level.DiscussionThe WATERLAND study aims to improve the early management of AP. Fluid resuscitation is an inexpensive treatment available in any hospital center worldwide. If a better evolution of pancreatitis is demonstrated in one of the treatment arms, it would have important repercussions in the management of this frequent disease.Trial registrationClinicalTrials.gov, NCT05781243. Registration date on January 4, 2023. EudraCT number 2023–000010-18, first posted March 23, 2023.

Polynitroxylated PEGylated hemoglobin protects pig brain neocortical gray and white matter after traumatic brain injury and hemorrhagic shock.

Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as an anti-inflammatory carbon monoxide donor. We determined whether small volume transfusion of hyperoncotic PNPH is neuroprotective in a porcine model of traumatic brain injury (TBI) with and without accompanying hemorrhagic shock (HS). TBI was produced by controlled cortical impact over the frontal lobe of anesthetized juvenile pigs. Hemorrhagic shock was induced starting 5 min after TBI by 30 ml/kg blood withdrawal. At 120 min after TBI, pigs were resuscitated with 60 ml/kg lactated Ringer's (LR) or 10 or 20 ml/kg PNPH. Mean arterial pressure recovered to approximately 100 mmHg in all groups. A significant amount of PNPH was retained in the plasma over the first day of recovery. At 4 days of recovery in the LR-resuscitated group, the volume of frontal lobe subcortical white matter ipsilateral to the injury was 26.2 ± 7.6% smaller than homotypic contralateral volume, whereas this white matter loss was only 8.6 ± 12.0% with 20-ml/kg PNPH resuscitation. Amyloid precursor protein punctate accumulation, a marker of axonopathy, increased in ipsilateral subcortical white matter by 132 ± 71% after LR resuscitation, whereas the changes after 10 ml/kg (36 ± 41%) and 20 ml/kg (26 ± 15%) PNPH resuscitation were not significantly different from controls. The number of cortical neuron long dendrites enriched in microtubules (length >50 microns) decreased in neocortex by 41 ± 24% after LR resuscitation but was not significantly changed after PNPH resuscitation. The perilesion microglia density increased by 45 ± 24% after LR resuscitation but was unchanged after 20 ml/kg PNPH resuscitation (4 ± 18%). Furthermore, the number with an activated morphology was attenuated by 30 ± 10%. In TBI pigs without HS followed 2 h later by infusion of 10 ml/kg LR or PNPH, PNPH remained neuroprotective. These results in a gyrencephalic brain show that resuscitation from TBI + HS with PNPH protects neocortical gray matter, including dendritic microstructure, and white matter axons and myelin. This neuroprotective effect persists with TBI alone, indicating brain-targeting benefits independent of blood pressure restoration.

Decay-Accelerating Factor Creates an Organ-Protective Phenotype after Hemorrhage in Conscious Rats.

Preclinical and clinical studies have shown that traumatic hemorrhage (TH) induces early complement cascade activation, leading to inflammation-associated multiple-organ dysfunction syndrome (MODS). Several previous studies have demonstrated the beneficial effects of complement inhibition in anesthetized (unconscious) animal models of hemorrhage. Anesthetic agents profoundly affect the immune response, microcirculation response, and coagulation patterns and thereby may confound the TH research data acquired. However, no studies have addressed the effect of complement inhibition on inflammation-driven MODS in a conscious model of hemorrhage. This study investigated whether early administration of decay-accelerating factor (CD55/DAF, a complement C3/C5 inhibitor) alleviates hemorrhage-induced organ damage and how DAF modulates hemorrhage-induced organ damage. DAF was administered to unanesthetized male Sprague Dawley rats subjected to pressure-controlled hemorrhage followed by a prolonged (4 h) hypotensive resuscitation with or without lactated Ringer's (LR). We assessed DAF effects on organ protection, tissue levels of complement synthesis and activation, T lymphocyte infiltration, fluid resuscitation requirements, and metabolic acidosis. Hemorrhage with (HR) or without (H) LR resuscitation resulted in significantly increased C3, C5a, and C5b-9 deposition in the lung and intestinal tissues. HR rats had significantly higher tissue levels of complement activation/deposition (particularly C5a and C5b-9 in the lung tissues), a higher but not significant amount of C3 and C5b-9 pulmonary microvascular deposition, and relatively severe injury in the lung and intestinal tissues compared to H rats. DAF treatment significantly reduced tissue C5b-9 formation and C3 deposition in the H or HR rats and decreased tissue levels of C5a and C3 mRNA in the HR rats. This treatment prevented the injury of these organs, improved metabolic acidosis, reduced fluid resuscitation requirements, and decreased T-cell infiltration in lung tissues. These findings suggest that DAF has the potential as an organ-protective adjuvant treatment for TH during prolonged damage control resuscitation.

Multifaceted Benefit of Whole Blood Versus Lactated Ringer's Resuscitation After Traumatic Brain Injury and Hemorrhagic Shock in Mice.

Despite increasing use in hemorrhagic shock (HS), whole blood (WB) resuscitation for polytrauma with traumatic brain injury (TBI) is largely unexplored. Current TBI guidelines recommend crystalloid for prehospital resuscitation. Although WB outperforms lactated Ringer's (LR) in increasing mean arterial pressure (MAP) in TBI + HS models, effects on brain tissue oxygenation (PbtO2), and optimal MAP remain undefined. C57BL/6 mice (n = 72) underwent controlled cortical impact followed by HS (MAP = 25-27mmHg). Ipsilateral hippocampal PbtO2 (n = 40) was measured by microelectrode. Mice were assigned to four groups (n = 18/group) for "prehospital" resuscitation (90min) with LR or autologous WB, and target MAPs of 60 or 70mmHg (LR60, WB60, LR70, WB70). Additional LR (10ml/kg) was bolused every 5min for MAP below target. LR requirements in WB60 (7.2 ± 5.0mL/kg) and WB70 (28.3 ± 9.6mL/kg) were markedly lower than in LR60 (132.8 ± 5.8mL/kg) or LR70 (152.2 ± 4.8mL/kg; all p < 0.001). WB70 MAP (72.5 ± 2.9mmHg) was higher than LR70 (59.8 ± 4.0mmHg, p < 0.001). WB60 MAP (68.7 ± 4.6mmHg) was higher than LR60 (53.5 ± 3.2mmHg, p < 0.001). PbtO2 was higher in WB60 (43.8 ± 11.6mmHg) vs either LR60 (25.9 ± 13.0mmHg, p = 0.04) or LR70 (24.1 ± 8.1mmHg, p = 0.001). PbtO2 in WB70 (40.7 ± 8.8mmHg) was higher than in LR70 (p = 0.007). Despite higher MAP in WB70 vs WB60 (p = .002), PbtO2 was similar. WB resuscitation after TBI + HS results in robust improvements in brain oxygenation while minimizing fluid volume when compared to standard LR resuscitation. WB resuscitation may allow for a lower prehospital MAP without compromising brain oxygenation when compared to LR resuscitation. Further studies evaluating the effects of these physiologic benefits on outcome after TBI with HS are warranted, to eventually inform clinical trials.

Direct Peritoneal Resuscitation Improves Survival in a Murine Model of Combined Hemorrhage and Burn Injury.

Combined burn injury and hemorrhagic shock are a common cause of injury in wounded warfighters. Current protocols for resuscitation for isolated burn injury and isolated hemorrhagic shock are well defined, but the optimal strategy for combined injury is not fully established. Direct peritoneal resuscitation (DPR) has been shown to improve survival in rats after hemorrhagic shock, but its role in a combined burn/hemorrhage injury is unknown. We hypothesized that DPR would improve survival in mice subjected to combined burn injury and hemorrhage. Male C57/BL6J mice aged 8weeks were subjected to a 7-second 30% total body surface area scald in a 90°C water bath. Following the scald, mice received DPR with 1.5mL normal saline or 1.5mL peritoneal dialysis solution (Delflex). Control mice received no peritoneal solution. Mice underwent a controlled hemorrhage shock via femoral artery cannulation to a systolic blood pressure of 25mmHg for 30minutes. Mice were then resuscitated to a target blood pressure with either lactated Ringer's (LR) or a 1:1 ratio of packed red blood cells (pRBCs) and fresh frozen plasma (FFP). Mice were observed for 24 hours following injury. Median survival time for mice with no DPR was 1.47hours in combination with intravascular LR resuscitation and 2.08 hours with 1:1 pRBC:FFP. Median survival time significantly improved with the addition of intraperitoneal normal saline or Delflex. Mice that received DPR followed by 1:1 pRBC:FFP required less intravascular volume than mice that received DPR with LR, pRBC:FFP alone, and LR alone. Intraperitoneal Delflex was associated with higher levels of tumor necrosis factor alpha and macrophage inflammatory protein 1 alpha and lower levels of interleukin 10 and intestinal fatty acid binding protein. Intraperitoneal normal saline resulted in less lung injury 1hour postresuscitation, but increased to similar severity of Delflex at 4hours. After a combined burn injury and hemorrhage, DPR leads to increased survival in mice. Survival was similar with the use of normal saline or Delflex. DPR with normal saline reduced the inflammatory response seen with Delflex and delayed the progression of acute lung injury. DPR may be a valuable strategy in the treatment of patients with combined burn injury and hemorrhage.

Pragmatic Pediatric Trial of Balanced Versus Normal Saline Fluid in Sepsis: The PRoMPT BOLUS Randomized Controlled Trial Pilot Feasibility Study.

Resuscitation with crystalloid fluid is a cornerstone of pediatric septic shock treatment. However, the optimal type of crystalloid fluid is unknown. We aimed to determine the feasibility of conducting a pragmatic randomized trial to compare balanced (lactated Ringer's [LR]) with 0.9% normal saline (NS) fluid resuscitation in children with suspected septic shock. Open-label pragmatic randomized controlled trial at a single academic children's hospital from January to August 2018. Eligible patients were>6 months to<18 years old who were treated in the emergency department for suspected septic shock, operationalized as blood culture, parenteral antibiotics, and fluid resuscitation for abnormal perfusion. Screening, enrollment, and randomization were carried out by the clinical team as part of routine care. Patients were randomized to receive either LR or NS for up to 48 hours following randomization. Other than fluid type, all treatment decisions were at the clinical team's discretion. Feasibility outcomes included proportion of eligible patients enrolled, acceptability of enrollment via the U.S. federal exception from informed consent (EFIC) regulations, and adherence to randomized study fluid administration. Of 59 eligible patients, 50 (85%) were enrolled and randomized. Twenty-four were randomized to LR and 26 to NS. Only one (2%) of 44 patients enrolled using EFIC withdrew before study completion. Total median (interquartile range [IQR]) crystalloid fluid volume received during the intervention window was 107 (60 to 155) mL/kg and 98 (63 to 128) mL/kg in the LR and NS arms, respectively (p=0.50). Patients randomized to LR received a median (IQR) of only 20% (13 to 32) of all study fluid as NS compared to 99% (64% to 100%) of study fluid as NS in the NS arm (absolute difference= 79%, 95% CI= 48% to 85%). A pragmatic study design proved feasible to study comparative effectiveness of LR versus NS fluid resuscitation for pediatric septic shock.

Safety of resuscitation with hydroxyethyl starch 130/0.4 in hemorrhagic shock rats

Objective: To evaluate the safety of resuscitation with hydroxyethyl starch 130/0.4 during the early stage of hemorrhagic shock. Methods: Total of 120 healthy male SD rats of 2 to 3 months of age were selected as the study sample (weighed from 250 to 290 g), all the rats were numbered by staining.After that, the rats were divided into 12 groups by using random number table method: sham group (S group), no liquid resuscitation group (NF group), lactated Ringer's resuscitation group (LR group), HES resuscitation group (HES group). At the same time, the LR and HES resuscitation groups were divided into five subgroups with a concentration of 7.5, 15, 30, 60 and 90 ml·kg(-1)·h(-1,) respectively.The model of uncontrollable hemorrhagic shock was created by the method of exsanguination plus tail-cuffing.Fluid resuscitation was started 30 minutes after the exsanguination and continued for 60 min after transfusion for 15 min.The observation was continued for 330 min.At the end of observation, all rats were sacrificed and blood was collected from the rats to determine the thromboelastograms and the maximum amplitude and related parameters, as well as platelet counts, blood urea nitrogen, creatinine, urinary and renal injury molecules, and neutrophil gelatinase-associated apolipoprotein levels.Rat lung tissue specimens were collected and wet weights of the right lung and dry weights after drying were measured.The data were compared by using one-way analysis of variance (ANOVA), LSD-t test or Dunnett-t test. Results: ANOVA analysis showed that there was no significant difference in mean artery pressure (MAP) values between groups at the beginning of fluid resuscitation (F=0.934, P=0.245). At the end of fluid resuscitation, the MAP of HES90 group was (40±9) mmHg, which was lower than that in other groups.Compared with other groups, the HES90 group had higher blood loss and blood transfusion rate.There was no significant differences in platelet counts between the HES group and the LR group at 330 min (t=0.987, P>0.05), but the maximal amplitude (MA) of the thrombelastogram (TEG) was lower in the HES90 group than that in the S group (t=2.354, P<0.05). No significant difference was detected in blood urea nitrogen and serum creatinine levels between the HES, LR group and the S group (t=1.098, 0.895, both P>0.05). The total amount of urinary kidney injury molecule 1 (Kim-1) in the HES90 and NF groups increased, neutrophil gelatinase-associated apolipoprotein (NGAL) concentration and urinary NGAL levels were significantly higher than those in other groups, and the difference were statistically significant (t=3.532-11.209, all P<0.05). Conclusion: Small to moderate doses of HES130/0.4 during hemorrhagic shock is more effective and safer than the same dose of LR.

Fibrinogen concentrate administration inhibits endogenous fibrinogen synthesis in pigs after traumatic hemorrhage.

Fibrinogen plays a central role in coagulation and falls to critical levels early after trauma. Administration of fibrinogen concentrate (FC) to improve hemostasis after severe bleeding seems beneficial, but it is unclear whether its use introduces excessive fibrinogen with a potential risk of thrombosis. This study investigated changes of endogenous fibrinogen metabolism from FC administration following traumatic hemorrhage in pigs. Anesthetized, instrumented pigs were randomized into lactated Ringer's (LR) solution only and LR plus FC groups (n = 7 each). Femur fracture of each pig's left leg was followed by hemorrhage of 60% total blood volume and resuscitation with LR (3× bled volume, LR group) or LR plus FC at 250 mg/kg (LR-FC group). Afterward, a constant infusion of stable isotopes 1-C-phenylalanine (phe, 6 hours) and d5-phe (3 hours) was performed with hourly blood sampling and subsequent gas chromatography-mass spectrometry analysis to quantify fibrinogen synthesis and breakdown rates, respectively. Blood gas and coagulation indices (thromboelastography) were measured on intermittent blood samples, and hemodynamics was continuously monitored. Animals were euthanized after the 6-hour isotope period. Mean arterial pressure decreased by 50% after hemorrhage but improved after LR resuscitation in both groups. Hemorrhage and LR resuscitation reduced total protein, hematocrit, fibrinogen, and platelets to 50% of baseline values. Moreover, hemorrhage and resuscitation decreased fibrinogen concentration (207 ± 6 vs. 132 ± 7 mg/dL) and clot strength (72 ± 2 vs. 63 ± 2 mm) in both groups (p < 0.05). FC administration restored plasma fibrinogen concentrations and clot strength within 15 minutes, while no changes occurred in the LR group. Fibrinogen synthesis rates in the LR-FC group (1.3 ± 0.2 mg/kg/h) decreased versus the LR group (3.1 ± 0.5; p < 0.05), whereas fibrinogen breakdown rates were similar. Our data suggest an effective feedback mechanism that regulates host fibrinogen availability and thereby suggests that acute thrombosis from FC administration is an unlikely risk.

Resveratrol Rescues Kidney Mitochondrial Function Following Hemorrhagic Shock.

Hemorrhagic shock may contribute to acute kidney injury (AKI) by profoundly altering renal mitochondrial function. Resveratrol (RSV), a naturally occurring sirtuin 1 (SIRT1) activator, has been shown to promote mitochondrial function and reduce oxidative damage in a variety of aging-related disease states. We hypothesized that RSV treatment during resuscitation would ameliorate kidney mitochondrial dysfunction and decrease oxidative damage following hemorrhagic shock. Using a decompensated hemorrhagic shock model, male Long-Evans rats (n = 6 per group) were killed prior to hemorrhage (sham), at severe shock, and following either lactated Ringer's (LR) resuscitation or LR + RSV resuscitation (RSV: 30 mg/kg). At each time point, blood samples were assayed for arterial blood gases, lactate, blood urea nitrogen, and serum creatinine. Mitochondria were also isolated from kidney samples in order to assess individual electron transport complexes (complexes I, II, and IV) using high-resolution respirometry. Total mitochondria reactive oxygen species were measured using fluorometry, and lipid peroxidation was assessed by measuring 4-hydroxynonenal by Western blot. Quantitative polymerase chain reaction was used quantify mRNA from peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α) SIRT1, and proteins known to mitigate oxidative damage and promote mitochondrial biogenesis. Resveratrol supplementation during resuscitation restored mitochondrial respiratory capacity and decreased mitochondrial reactive oxygen species and lipid peroxidation. Compared with standard LR resuscitation, RSV treatment significantly increased SIRT1 and PGC1-α expression and significantly increased both superoxide dismutase 2 and catalase expression. Although RSV was associated with decreased lactate production, pH, blood urea nitrogen, and serum creatinine values did not differ between resuscitation strategies. Resuscitation with RSV significantly restored renal mitochondrial function and decreased oxidative damage following hemorrhagic shock.

Fresh Frozen Plasma Lessens Pulmonary Endothelial Inflammation and Hyperpermeability After Hemorrhagic Shock and Is Associated With Loss of Syndecan 1

We have recently demonstrated that injured patients in hemorrhagic shock shed syndecan 1 and that the early use of fresh frozen plasma (FFP) in these patients is correlated with improved clinical outcomes. As the lungs are frequently injured after trauma, we hypothesized that hemorrhagic shock-induced shedding of syndecan 1 exposes the underlying pulmonary vascular endothelium to injury resulting in inflammation and hyperpermeability and that these effects would be mitigated by FFP. In vitro, pulmonary endothelial permeability, endothelial monolayer flux, transendothelial electrical resistance, and leukocyte-endothelial binding were measured in pulmonary endothelial cells after incubation with equal volumes of FFP or lactated Ringer's (LR). In vivo, using a coagulopathic mouse model of trauma and hemorrhagic shock, pulmonary hyperpermeability, neutrophil infiltration, and syndecan 1 expression and systemic shedding were assessed after 3 h of resuscitation with either 1× FFP or 3× LR and compared with shock alone and shams. In vitro, endothelial permeability and flux were decreased, transendothelial electrical resistance was increased, and leukocyte-endothelial binding was inhibited by FFP compared with LR-treated endothelial cells. In vivo, hemorrhagic shock was associated with systemic shedding of syndecan 1, which correlated with decreased pulmonary syndecan 1 and increased pulmonary vascular hyperpermeability and inflammation. Fresh frozen plasma resuscitation, compared with LR resuscitation, abrogated these injurious effects. After hemorrhagic shock, FFP resuscitation inhibits endothelial cell hyperpermeability and inflammation and restores pulmonary syndecan 1 expression. Modulation of pulmonary syndecan 1 expression may mechanistically contribute to the beneficial effects FFP.

Comparisons of lactated Ringer’s and Hextend resuscitation on hemodynamics and coagulation following femur injury and severe hemorrhage in pigs

This study compared coagulation function after resuscitation with Hextend and lactated Ringer's (LR) solution in pigs with tissue injury and hemorrhagic shock. Pigs were randomized into control (n = 7 each), LR, and Hextend groups. Femur fracture was induced using the captive bolt stunner at midshaft of the pigs' left legs, followed by hemorrhage of 60% total blood volume and resuscitation with either Hextend (equal to bled volume) or LR to reach the same mean arterial pressure. Pigs in the control group were not bled or resuscitated. Hemodynamics was monitored hourly for 6 hours. Blood samples were taken at baseline (BL), after hemorrhage, 15 minutes, 3 hours, and 6 hours after resuscitation for blood and coagulation measurements. Mean arterial pressure decreased to 50% of BL by the 60% hemorrhage but returned to near BL within 1 hour after LR or Hextend resuscitation. Heart rate was increased (from 91 ± 4 beats per minute to 214 ± 20 beats per minute) by hemorrhage and decreased after resuscitation but remained elevated above BL in both groups. Resuscitation with Hextend (42 mL/kg) or LR (118 ± 3 mL/kg) reduced hematocrit, total protein, fibrinogen, and platelet counts, with greater decreases shown in the Hextend group. Clot strength was lower but returned to BL by 3 hours in the LR group, whereas it remained reduced for the 6-hour period after Hextend. The overall clotting capacity after LR was decreased after hemorrhage and resuscitation but returned to BL by 3 hours, whereas it remained low after Hextend for the 6-hour experiment period. After traumatic hemorrhage, coagulation function was restored within 6 hours with LR resuscitation but not with Hextend. The lack of recovery after Hextend is likely caused by greater hemodilution and possible effects of starches on coagulation substrates and further documents the need to restrict the use of high-molecular-weight starch in resuscitation fluids for bleeding casualties.

Ulinastatin Suppresses Burn-Induced Lipid Peroxidation and Reduces Fluid Requirements in a Swine Model

Objective. Lipid peroxidation plays a critical role in burn-induced plasma leakage, and ulinastatin has been reported to reduce lipid peroxidation in various models. This study aims to examine whether ulinastatin reduces fluid requirements through inhibition of lipid peroxidation in a swine burn model. Methods. Forty miniature swine were subjected to 40% TBSA burns and were randomly allocated to the following four groups: immediate lactated Ringer's resuscitation (ILR), immediate LR containing ulinastatin (ILR/ULI), delayed LR resuscitation (DLR), and delayed LR containing ulinastatin (DLR/ULI). Hemodynamic variables, net fluid accumulation, and plasma thiobarbituric acid reactive substances (TBARS) concentrations were measured. Heart, liver, lung, skeletal muscle, and ileum were harvested at 48 hours after burn for evaluation of TBARS concentrations, activities of antioxidant enzymes, and tissue water content. Results. Ulinastatin significantly reduced pulmonary vascular permeability index (PVPI) and extravascular lung water index (ELWI), net fluid accumulation, and water content of heart, lung, and ileum in both immediate or delayed resuscitation groups. Furthermore, ulinastatin infusion significantly reduced plasma and tissue concentrations of TBARS in both immediate or delayed resuscitation groups. Conclusions. These results indicate that ulinastatin can reduce fluid requirements through inhibition of lipid peroxidation.

531

Introduction: Polynitroxylated pegylated hemoglobin (PNPH), a bovine hemoglobin decorated with niroxide and polyethylene glycol moieties, demonstrated neuroprotection in vitro and in vivo vs. Lactated Ringer’s (LR) in experimental TBI plus hemorrhagic shock (HS). Hypothesis: We hypothesized that resuscitation with PNPH will reduce intracranial pressure (ICP) and brain edema vs. LR in experimental TBI+HS. Methods: C57/BL6 male mice (n=20) were anesthetized with isoflurane and underwent controlled cortical impact (CCI) followed by HS to mean arterial pressure (MAP) of 25-27mm Hg for 35 min. Mice (n=10/group) were then resuscitated with an initial bolus of 20ml/kg of either 4% PNPH or LR followed by 10ml/kg boluses every 5 min for MAP <70 mm Hg for 90min, mimicking pre-hospital care. Shed blood was then re-infused. ICP was monitored for the duration of the model after which the mice were sacrificed and percent brain water was quantified (wet/dry weight). Results: Mice resuscitated with PNPH vs. LR required less fluid for resuscitation (26 ± 0 vs. 167 ± 10.7 ml/kg, P<0.001) and had a higher MAP at the end of the pre-hospital phase (78 ± 4.7 vs. 60.1 ± 8.8 mm Hg, P<0.001). The PNPH mice required only the initial resuscitation bolus of 20 ml/kg, while the LR mice required multiple boluses during resuscitation for MAP <70mmHg. PNPH-treated mice also had a lower peak ICP (14.5 ± 3.1 vs 19.6 ± 3.5 mm Hg, P=0.003) and higher cerebral perfusion pressure (CPP) at the end of the pre-hospital phase (67.1 vs. 45 mmHg, P<0.001). The PNPH mice also had a reduced %brain water in the hemisphere ipsilateral to injury vs. LR (80.3 ± 0.1 vs. 80.9 ± 0.1%). Hemoglobin and lactate levels were not different between the groups at any time point. Methemoglobin levels were mildly elevated in the PNPH group in the pre-hospital and hospital phase (P<0.001). Conclusions: Resuscitation with PNPH dramatically lowers fluid requirements, improves ICP and CPP and reduces brain edema vs. LR resuscitation. Despite severe HS, PNPH-treated mice required only a single bolus of fluid and were adequately resuscitated while the LR mice were refractory to resuscitation. Our data support ongoing pre-clinical development of this novel resuscitation fluid for TBI resuscitation.

Different recovery profiles of coagulation factors, thrombin generation, and coagulation function after hemorrhagic shock in pigs

Hemorrhagic shock contributes to coagulopathy after trauma. We investigated daily changes of coagulation components and coagulation function for 5 days in hemorrhaged and resuscitated pigs. Fourteen pigs were randomized into the sham control (C) and the hemorrhage and lactated Ringer's resuscitation (H-LR) groups. On day 1, hemorrhage was induced in the H-LR group by bleeding 35% of the total blood volume, followed by LR resuscitation at three times the bled volume. Pigs in the C group were not hemorrhaged or resuscitated. Hemodynamics and coagulation were measured daily after H-LR on day 1 to day 5. No changes in hemodynamics and coagulation function occurred in C. Hemorrhage decreased mean arterial pressure and increased heart rate. LR resuscitation corrected these changes within 2 hours. Compared with the baseline values (BL) on day 1, fibrinogen levels were decreased to 76% ± 6% by H-LR on day 1, increased to 217% ± 16% on day 2, and remained increased thereafter; platelet counts were decreased to 63% ± 5% by H-LR on day 1 and remained lower on days 2 and 3 but returned to BL by days 4 and 5 (all p < 0.05). Thrombin generation was decreased by H-LR on days 1 and 2 but then increased to above BL on days 4 and 5. Coagulation factor levels were decreased by H-LR on day 1 but returned to BL on day 3 except for factor XIII. Clot strength was decreased by H-LR on day 1 and returned to BL by day 2. Clot rapidity did not change on day 1 but was decreased on days 2 and 3 and returned to BL on days 4 and 5. Hemorrhage and resuscitation reduced coagulation components and compromised coagulation function, which showed different recovery profiles over the 5-day study period.

Pyruvate-Fortified Fluid Resuscitation Improves Hemodynamic Stability While Suppressing Systemic Inflammation and Myocardial Oxidative Stress After Hemorrhagic Shock

To determine whether controlled resuscitation with pyruvate-fortified Ringer's (PR) solution vs. conventional lactate Ringer's (LR) more effectively stabilizes mean arterial pressure (MAP) and suppresses myocardial inflammation postresuscitation. Goats were hemorrhaged (255 +/- 22 ml) to lower MAP to 48 +/- 1 mmHg. Next, the right femoral vessels were occluded for 90 min to model tourniquet application. Beginning at 30 min occlusion, LR or PR was infused i.v. at 10 ml/min for 90 min. The femoral occlusions were released at 60 min infusion. At 4 h postocclusion, MAP (mmHg) was increased in PR (59 +/- 4) vs. LR (47 +/- 3) resuscitated goats (p < 0.05). PR also more effectively augmented circulating HCO3 and total base excess. Nitrosative stress, detected in myocardium 4 h after LR resuscitation, was suppressed by PR. Finally, PR prevented the increase in circulating neutrophils that accompanied LR resuscitation. Relative to LR, resuscitation with PR more effectively stabilized MAP, suppressed myocardial nitrosative stress and minimized systemic inflammation after hemorrhagic shock with hindlimb ischemia-reperfusion.

Resuscitation With Fresh Whole Blood Ameliorates the Inflammatory Response After Hemorrhagic Shock

Hemorrhagic shock is the leading cause of potentially preventable death after traumatic injury. Hemorrhage and subsequent resuscitation may result in a dysfunctional systemic inflammatory response and multisystem organ failure, leading to delayed mortality. Clinical evidence supports improved survival and reduced morbidity when fresh blood products are used as resuscitation strategies. We hypothesized that the transfusion of fresh whole blood (FWB) attenuates systemic inflammation and reduces organ injury when compared with conventional crystalloid resuscitation after hemorrhagic shock. Male mice underwent femoral artery cannulation and hemorrhage to a systolic blood pressure of 25 mm Hg +/- 5 mm Hg. After 60 minutes, the mice were resuscitated with either FWB or lactated Ringer's solution (LR). Mice were decannulated and killed at intervals for tissue histology, serum cytokine analysis, and vascular permeability studies. Separate groups of mice were followed for survival studies. When compared with FWB, mice resuscitated with LR required increased resuscitation fluid volume to reach goal systolic blood pressure. When compared with sham or FWB-resuscitated mice, LR resuscitation resulted in increased serum cytokine levels of macrophage inflammatory protein-1alpha, interleukin-6, interleukin-10, macrophage-derived chemokine, KC, and granulocyte macrophage colony stimulating factor as well as increased lung injury and pulmonary capillary permeability. No survival differences were seen between animals resuscitated with LR or FWB. Resuscitation with LR results in increased systemic inflammation, vascular permeability, and lung injury after hemorrhagic shock. Resuscitation with FWB attenuates the inflammation and lung injury seen with crystalloid resuscitation. These findings suggest that resuscitation strategies using fresh blood products potentially reduce systemic inflammation and organ injury after hemorrhagic shock.

Comparison of plasma volume assessments following hemorrhagic shock in pigs

Indocyanine green (ICG) dye is frequently used for plasma volume (PV) measurements. However, the accuracy of ICG measurements following severe hemorrhage (H) is unclear due to limited knowledge of its metabolism when liver function is altered by H. This study assessed changes of PV in pigs after H and compared the ICG results with Hct and total protein. Sixteen pigs were divided into controls (n=4) or H with either LR (n=6) or saline (n=6) resuscitation. H was induced by withdrawing 60% of the estimated blood volume from the femoral artery, followed by resuscitation with LR or saline. ICG (25mg/10ml) was injected iv to assess PV at baseline and 6 h after H and resuscitation. Blood samples were taken before each injection for blood gas and chemistry. PV was also calculated based on the changes of Hct and total protein for comparison. There were no changes in PV in controls throughout the study. At 6 h following H and resuscitation, PV by ICG was 121±3% and 120±4% of baseline in the LR and saline groups, respectively. PV calculated by changes of Hct showed 119±1% and 119±2% of baseline in these groups, respectively. Similarly, PV calculated by changes of total protein was 121±6% and 124±3 % of baseline in these groups, respectively. In conclusion, these data suggest that following severe H, ICG remains a reliable method for measurements of PV.This work was supported by US Army Medical Research and Medical Command.

EFFECTS OF HEMORRHAGE AND LACTATED RINGER'S RESUSCITATION ON COAGULATION AND FIBRINOGEN METABOLISM IN SWINE

Hemorrhagic coagulopathy is a significant complication after traumatic injury, and much of the underlying mechanism remains unclear. We investigated the changes in fibrinogen metabolism and coagulation after a moderate hemorrhage and resuscitation. Pigs of either sex (weight, 40.9+/-0.8 kg) were anesthetized and instrumented with arterial and venous catheters and a thermodilution cardiac output catheter. Pigs were randomized into control (C; n=6), hemorrhage (H; n=6), and hemorrhage and resuscitation (H-LR; n=6) groups. Hemorrhage was induced by bleeding 35% of total blood volume for 30 min in H and H-LR groups. Resuscitation in H-LR group was performed using lactated Ringer's solution (LR) at 3 times the bled volume for 30 min. Fibrinogen metabolism was quantified using a primed constant infusion of 1-13C-phenylalanine (6 h) and d5-phenylalanine (4 h) and subsequent analysis by gas chromatograph-mass spectrometry, together with measurements of hemodynamics (hourly) and coagulation by thromboelastography (at baseline and 4 h after hemorrhage and resuscitation). Hemorrhage caused decreases in arterial pH and base excess, and an increase in arterial lactate content. Fluid resuscitation corrected these changes toward normal levels. Fibrinogen level was unchanged in C and decreased to 76%+/-4% in H and to 73%+/-3% in H-LR (both P<0.05, compared with baseline) after hemorrhage and resuscitation. Fibrinogen breakdown was increased from 3.0+/-0.4 mg kg-1 h-1 in C to 5.4+/-0.6 mg kg-1 h-1 in H and to 5.6+/-0.5 mg kg-1 h-1 in H-LR (both P<0.05, compared with control), but synthesis was unchanged. The clotting reaction time was unchanged in C and shortened to 93%+/-3% in H and to 91%+/-1% in H-LR (both P<0.05, compared with baseline). We conclude that hemorrhagic shock caused accelerated fibrinogen breakdown and coagulation. The LR resuscitation reduced tissue hypoxia indexes but did not affect the changes in fibrinogen metabolism and coagulation from hemorrhage. Thus, effective treatment of hemorrhage should include combining standard-of-care resuscitation with interventions to correct alterations in coagulation.

Fluid Resuscitation Increases Inflammatory Gene Transcription After Traumatic Injury

The debate continues over type and quantity of fluid to administer for resuscitation after traumatic injury. This study aimed to examine effects of resuscitation with lactated Ringer's (LR) and Hextend (HEX) on the inflammatory response after uncontrolled hemorrhagic shock (UHS). There were 38 swine randomized. Control swine were anesthetized and killed. Sham swine underwent laparotomy, splenectomy, and 2 hours of anesthesia. UHS swine received a grade V liver injury after laparotomy and splenectomy and were randomized to no fluid (NF) resuscitation or to blinded resuscitation 30 minutes after injury with LR or HEX. Fluids were infused as needed to maintain baseline blood pressure for 90 minutes. Lung tissue mRNA levels of interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF), and tumor necrosis factor alpha (TNF-alpha) were determined. Lung sections were examined for neutrophils (PMNs) sequestered within alveolar walls. All UHS animals survived and initial blood loss was similar between groups. Mean arterial pressures (MAPs) were similar for all UHS animals until resuscitation was initiated. MAPs of resuscitated animals remained similar and were significantly higher than MAPs of the NF animals. Sequestered PMNs were equally elevated in all UHS animals. Cytokine analysis showed increased IL-6, G-CSF, and TNF-alpha gene transcription in resuscitated swine compared with NF swine. LR and HEX resuscitated swine tissue mRNA levels showed no differences. Fluid resuscitation after solid organ injury and uncontrolled hemorrhage results in greater proinflammatory gene transcription than no resuscitation. LR and HEX resuscitation have equivalent effects on indices of inflammation in the lungs.

EFFECTS OF HEMORRHAGE AND LACTATED RINGER'S (LR) RESUSCITATION ON COAGULATION AND FIBRINOGEN METABOLISM IN PIGS

Objective: Hemorrhagic coagulopathy is a significant complication following traumatic injury and much of the underlying mechanism remains unclear. We investigated changes in fibrinogen metabolism and coagulation following a moderate hemorrhage and resuscitation. Methods: Pigs of either sex (40 ± 1 kg) were anesthetized and instrumented with arterial and venous catheters, and a thermodilution cardiac output (CO) catheter. Pigs were randomized into control (C; n = 6), hemorrhage (H, n = 6), and hemorrhage and resuscitation (H-LR, n = 6) groups. Hemorrhage was induced by bleeding 35% of total blood volume in 30 min in H and H-LR. Resuscitation was performed using LR at 3 times the bled volume over 30 min in H-LR. Fibrinogen metabolism was quantified using a primed constant infusion of 1-13C-phenylalanine (phe, 6h) and d5-phe (4h) and subsequent analysis by gas chromatograph mass spectrometry, together with measurements of hemodynamics and coagulation (by TEG). Results: Hemorrhage caused decreases in mean arterial pressure, CO, pH and BE, as well as an increase in lactate content. Resuscitation corrected these changes toward normal. Clotting R time was unchanged in C, and shortened to 93 ± 3% in H and to 91 ± 1% in H-LR (both p <0.05, compare to baseline). Fibrinogen level was unchanged in C and decreased to 76 ± 4% in H and to 73 ± 3% in H-LR (both p <0.05, compare to baseline). Fibrinogen breakdown was increased from 3.0 ± 0.4 mg/kg/h in C to 5.4 ± 0.6 mg/kg/h in H and to 5.6 ± 0.5 mg/kg/h in H-LR (both p <0.05, compare to control), but synthesis was unchanged. Conclusion: Hemorrhage shock caused accelerated fibrinogen breakdown and coagulation. LR resuscitation reduced tissue hypoxia indices but did not affect the changes in fibrinogen metabolism and coagulation from hemorrhage. Thus, effective treatment of hemorrhage should include combining standard of care resuscitation with interventions to correct alterations in coagulation.