Pressure-controlled Hemorrhagic Shock Research Articles

5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) improves lipid utilization but not survival following prolonged hemorrhagic shock

Hemorrhagic shock induces insulin resistance and cellular ATP depletion, the initial step for cell death and organ injury. AMPK is a fuel-sensing enzyme that maintains ATP production by facilitating glucose and fatty acid uptake and oxidation. Using a previously developed rat model of AKI induced by prolonged hemorrhagic shock, we tested the hypothesis that AICAR, an AMPK activator, can enhance fuel consumption, delay onset of AKI, and increase survival following prolonged hemorrhagic shock. Additionally, we hypothesized that AICAR requires 7.5% polyethylene glycol-20k (PEG) to elicit the catabolism effect, as PEG enhances the capillary perfusion in the absence of resuscitation, and thus, in theory, facilitates AICAR delivery into ischemic tissues. Rats were randomized into 4 groups (n=8/group) based on treatment: saline (vehicle), AICAR, PEG, and AICAR+PEG. Trauma was induced in (Inactin) anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhagic shock (MAP = 55 mmHg) for 45 minutes. Animals then received an intravenous injection of saline (vehicle), AICAR, PEG, or AICAR+PEG. MAP, blood gases, RBF, renal oxygen delivery, capillary perfusion and tissue oxygen levels in the skeletal muscle were monitored for another 2 hours. GFR and mortality were recorded at the end of the experiment. The saline group exhibited impaired tissue perfusion and GFR with 100% mortality. AICAR alone showed no effect on hemodynamics, renal function, or mortality (100%). PEG and AICAR+PEG similarly improved MAP, capillary perfusion in the skeletal muscle, renal blood flow, renal oxygen delivery, and GFR. Compared to PEG, free FA levels were further decreased in AICAR+PEG, which was concomitant with elevated lactate levels and blunted oxygen partial pressure in the skeletal muscle despite the improved capillary perfusion. The mortality was 12.5% in PEG and 50% in AICAR+PEG. Our results suggest that AICAR+PEG does enhance fuel utilization but fails to further improve the tolerance to hemorrhagic shock, possibly due to a shift of oxygen consumption from vital organs to peripheral tissue. Importantly, the catabolism effect of AICAR relies on suffcient tissue perfusion, suggesting that PEG might be potentially considered as a routine therapeutic tool to optimize drug delivery during hemorrhagic shock. DoD disclaimer: The views expressed in this article are those of the author(s) and do not reflect the offcial policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government. Funding: This study was funded by US Army Medical Research and Development Command. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alleviating renal injury and mitochondrial dysfunction following hemorrhagic shock

Resuscitation with polydatin (PD) has been shown to alleviate mitochondrial damage and ischemia reperfusion injury following hemorrhagic shock. However, whether PD has the same beneficial effect under prolonged hemorrhagic shock with delayed or limited resuscitation is unclear. Using a previously developed rat model of acute kidney injury (AKI) induced by prolonged hemorrhagic shock, we tested the hypothesis that PD preserves renal mitochondrial function, delays the onset of AKI, and decreases mortality following prolonged hemorrhagic shock without full resuscitation. Additionally, we determined whether polyethylene glycol-20k (PEG) can enhance the beneficial effects of PD via improved capillary perfusion and PD delivery into the ischemic tissues. Rats were randomized into 4 groups (n=8/group) based on treatment: saline (vehicle), PD, PEG, and PD+PEG. Trauma was induced in Inactin anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhagic shock (mean arterial pressure (MAP) = 55 mmHg) for 45 minutes. Animals then received an intravenous injection of saline (vehicle), PD, PEG, or PD+PEG. MAP, blood gases, hematocrit, renal blood flow (RBF), and oxygen delivery were monitored for another 2 hours. Glomerular filtration rate (GFR), urinary sodium uptake, renal mitochondrial function, and mortality were recorded at the end of the experiment. The saline group exhibited AKI, renal mitochondrial dysfunction, and 100% mortality. PD alone showed no effect on mitochondrial function, renal injury, or mortality (100%). PD+PEG restored both complex II and IV activities, while PEG improved mitochondrial complex II function but showed no effect on complex IV. Nevertheless, PEG and PD+PEG similarly improved MAP, renal blood flow, renal oxygen delivery, GFR, sodium uptake, and mortality (PEG: 12.5%; PD+PEG: 0%). Our results suggest that polydatin requires suffcient tissue perfusion to exert its beneficial effects on the mitochondrial function of hemorrhagic shock animals. We used clinically meaningful endpoints to demonstrate the utility of PEG as a therapeutic tool for optimizing drug delivery to treat hemorrhagic shock. DoD disclaimer: The views expressed in this article are those of the author(s) and do not reflect the offcial policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government. Funding: This study was funded by US Army Medical Research and Development Command. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CAN POLYETHYLENE GLYCOL-20K REPLACE ALBUMIN FOR PREHOSPITAL TREATMENT OF HEMORRHAGIC SHOCK WHEN FULL RESUSCITATION IS UNAVAILABLE?

A solution of high concentration albumin has been used for temporal volume expansion when timely resuscitation was unavailable after hemorrhagic shock. However, during prolonged hemorrhagic shock, cell edema and interstitial dehydration can occur and impede the volume expansion effect of albumin. Polyethylene glycol-20K (PEG) can establish an osmotic gradient from swollen cells to capillary lumens and thus facilitate capillary fluid shift and volume expansion. We hypothesized that with similar osmolality, 7.5% PEG elicits more rapid and profound compensatory responses after hemorrhagic shock than 25% albumin. Rats were randomized into three groups (n = 8/group) based on treatment: saline (vehicle), PEG (7.5%), and albumin (25%). Trauma was induced in anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhagic shock (MAP = 55 mm Hg) for 45 min. Animals then received an intravenous injection (0.3 mL/kg) of saline, PEG, or albumin. MAP, heart rate, blood gases, hematocrit, skeletal muscle capillary flow, renal blood flow, glomerular filtration rate, urinary flow, urinary sodium concentration, and mortality were monitored for another 2 hours. Polyethylene glycol-20K and albumin both improved MAP, renal and capillary blood flow, and renal oxygen delivery, and decreased hyperkalemia, hyperlactatemia, hematocrit, and mortality (saline: 100% PEG: 12.5%; albumin: 38%) over saline treatment. Compared with albumin, PEG had a more rapid decrease in hematocrit and more profound increases in MAP, diastolic pressure, renal blood flow, glomerular filtration rate, and urinary flow. These results suggest that PEG may be a better option than albumin for prolonged prehospital care of hemorrhagic shock.

Cigarette smoke exposure reduces hemorrhagic shock induced circulatory dysfunction in mice with attenuated glucocorticoid receptor function.

IntroductionWe previously showed that attenuated glucocorticoid receptor (GR) function in mice (GRdim/dim) aggravates systemic hypotension and impairs organ function during endotoxic shock. Hemorrhagic shock (HS) causes impaired organ perfusion, which leads to tissue hypoxia and inflammation with risk of organ failure. Lung co-morbidities like chronic obstructive pulmonary disease (COPD) can aggravate tissue hypoxia via alveolar hypoxia. The most common cause for COPD is cigarette smoke (CS) exposure. Therefore, we hypothesized that affecting GR function in mice (GRdim/dim) and pre-traumatic CS exposure would further impair hemodynamic stability and organ function after HS.MethodsAfter 3 weeks of CS exposure, anesthetized and mechanically ventilated GRdim/dim and GR+/+ mice underwent pressure-controlled HS for 1h via blood withdrawal (mean arterial pressure (MAP) 35mmHg), followed by 4h of resuscitation with re-transfusion of shed blood, colloid fluid infusion and, if necessary, continuous intravenous norepinephrine. Acid–base status and organ function were assessed together with metabolic pathways. Blood and organs were collected at the end of the experiment for analysis of cytokines, corticosterone level, and mitochondrial respiratory capacity. Data is presented as median and interquartile range.ResultsNor CS exposure neither attenuated GR function affected survival. Non-CS GRdim/dim mice had a higher need of norepinephrine to keep target hemodynamics compared to GR+/+ mice. In contrast, after CS exposure norepinephrine need did not differ significantly between GRdim/dim and GR+/+ mice. Non-CS GRdim/dim mice presented with a lower pH and increased blood lactate levels compared to GR+/+ mice, but not CS exposed mice. Also, higher plasma concentrations of some pro-inflammatory cytokines were observed in non-CS GRdim/dim compared to GR+/+ mice, but not in the CS group. With regards to metabolic measurements, CS exposure led to an increased lipolysis in GRdim/dim compared to GR+/+ mice, but not in non-CS exposed animals.ConclusionWhether less metabolic acidosis or increased lipolysis is the reason or the consequence for the trend towards lower catecholamine need in CS exposed GRdim/dim mice warrants further investigation.

Sulforaphane Exerts Beneficial Immunomodulatory Effects on Liver Tissue via a Nrf2 Pathway-Related Mechanism in a Murine Model of Hemorrhagic Shock and Resuscitation.

Our research explores the immunomodulatory effects of sulforaphane (SFN), a well-known nuclear factor erythroid 2-related factor 2 (Nrf2) pathway agonist, on the sterile inflammation of and ischemia-reperfusion injuries to the liver after hemorrhagic shock (HS) followed by resuscitation (R). Male C57/BL6 wild-type and transgenic ARE-luc mice were exposed to mean arterial pressure-controlled HS. Fluid resuscitation was performed after 90 min of HS, and SFN was administrated intraperitoneally after that. The animals were sacrificed at 6 h, 24 h, and 72 h after resuscitation, and their livers were extracted to perform H&E staining and myeloperoxidase (MPO) activity analysis. The Kupffer cells were isolated for cytokines profile measurements and Nrf2 immunofluorescence staining. Further, the ARE-luc mice were used to assess hepatic Nrf2 activity in vivo. We identified that SFN-activated Kupffer cells’ Nrf2 pathway and modulated its cytokines expression, including TNF-α, MCP-1, KC/CXCL1, IL-6, and IL-10. Furthermore, SFN mitigated liver ischemia-reperfusion injury, as evidenced by the downregulation of the Suzuki score and the enhanced hepatic Nrf2 activity. The in vivo SFN treatment decreased neutrophils infiltration, as shown by the decreased MPO levels. Our study shows that SFN can decrease HS/R-induced hepatic ischemia-reperfusion injury and modulate the activity of Kupffer cells via an Nrf2-dependent pathway.

Hemorrhagic Shock Induces a Rapid Transcriptomic Shift of the Immune Balance in Leukocytes after Experimental Multiple Injury.

The immune response following trauma represents a major driving force of organ dysfunction and poor outcome. Therefore, we investigated the influence of an additional hemorrhagic shock (HS) on the early posttraumatic immune dysbalance in the whole population of blood leukocytes. A well-established murine polytrauma (PT) model with or without an additional pressure-controlled HS (mean arterial pressure of 30 mmHg (±5 mmHg) for 60 mins, afterwards fluid resuscitation with balanced electrolyte solution four times the volume of blood drawn) was used. C57BL/6 mice were randomized into a control, PT, and PT + HS group with three animals in each group. Four hours after trauma, corresponding to three hours after induction of hemorrhage, RNA was isolated from all peripheral blood leukocytes, and a microarray analysis was performed. Enrichment analysis was conducted on selected genes strongly modulated by the HS. After additional HS in PT mice, the gene expression of pathways related to the innate immunity, such as IL-6 production, neutrophil chemotaxis, cell adhesion, and toll-like receptor signaling was upregulated, whereas pathways of the adaptive immune system, such as B- and T-cell activation as well as the MHC class II protein complex, were downregulated. These results demonstrate that an additional HS plays an important role in the immune dysregulation early after PT by shifting the balance to increased innate and reduced adaptive immune responses.

Effect of Polyethylene-glycolated Carboxyhemoglobin on Renal Microcirculation in a Rat Model of Hemorrhagic Shock.

Primary resuscitation fluid to treat hemorrhagic shock remains controversial. Use of hydroxyethyl starches raised concerns of acute kidney injury. Polyethylene-glycolated carboxyhemoglobin, which has carbon monoxide-releasing molecules and oxygen-carrying properties, was hypothesized to sustain cortical renal microcirculatory PO2 after hemorrhagic shock and reduce kidney injury. Anesthetized and ventilated rats (n = 42) were subjected to pressure-controlled hemorrhagic shock for 1 h. Renal cortical PO2 was measured in exposed kidneys using a phosphorescence quenching method. Rats were randomly assigned to six groups: polyethylene-glycolated carboxyhemoglobin 320 mg · kg, 6% hydroxyethyl starch (130/0.4) in Ringer's acetate, blood retransfusion, diluted blood retransfusion (~4 g · dl), nonresuscitated animals, and time control. Nitric oxide and heme oxygenase 1 levels were determined in plasma. Kidney immunohistochemistry (histologic scores of neutrophil gelatinase-associated lipocalin and tumor necrosis factor-α) and tubular histologic damages analyses were performed. Blood and diluted blood restored renal PO2 to 51 ± 5 mmHg (mean difference, -18; 95% CI, -26 to -11; P < 0.0001) and 47 ± 5 mmHg (mean difference, -23; 95% CI, -31 to -15; P < 0.0001), respectively, compared with 29 ± 8 mmHg for hydroxyethyl starch. No differences between polyethylene-glycolated carboxyhemoglobin and hydroxyethyl starch were observed (33 ± 7 mmHg vs. 29 ± 8 mmHg; mean difference, -5; 95% CI, -12 to 3; P = 0.387), but significantly less volume was administered (4.5 [3.3-6.2] vs. 8.5[7.7-11.4] ml; mean rank difference, 11.98; P = 0.387). Blood and diluted blood increased the plasma bioavailability of nitric oxide compared with hydroxyethyl starch (mean rank difference, -20.97; P = 0.004; and -17.13; P = 0.029, respectively). No changes in heme oxygenase 1 levels were observed. Polyethylene-glycolated carboxyhemoglobin limited tubular histologic damages compared with hydroxyethyl starch (mean rank difference, 60.12; P = 0.0012) with reduced neutrophil gelatinase-associated lipocalin (mean rank difference, 84.43; P < 0.0001) and tumor necrosis factor-α (mean rank difference, 49.67; P = 0.026) histologic scores. Polyethylene-glycolated carboxyhemoglobin resuscitation did not improve renal PO2 but limited tubular histologic damages and neutrophil gelatinase-associated lipocalin upregulation after hemorrhage compared with hydroxyethyl starch, whereas a lower volume was required to sustain macrocirculation.

Remote Intestinal Injury Early After Experimental Polytrauma and Hemorrhagic Shock.

Dysfunction of the gut-blood barrier plays an important role in many diseases, such as inflammatory bowel disease, hemorrhagic shock (HS), or burn injury. However, little is known about gut barrier dysfunction after hemodynamically instable polytrauma (PT). Therefore, we aimed to evaluate the effects of PT and HS on remote intestinal damage and barrier dysfunction, especially regarding the role of zonula occludens protein 1 (ZO-1) as an important tight junction protein.Male C57BL/6 mice were subjected to either PT (thorax trauma, closed head injury, soft tissue injury, and distal femoral fracture), 60 min of pressure-controlled HS (30 ± 5 mmHg), or PT+HS, or sham procedures.Animals of all trauma groups showed an increase in abdominal girth and dilation of the intestine during the experimental period, which was largest in the PT+HS group. Increased blood-tissue permeability to albumin (assessed by Evans blue dye) was found in the HS group. Experimental groups showed a slight increase in plasma concentration of intestinal fatty acid binding protein and some intestinal damage was histologically detectable. Of note, PT+HS animals revealed significantly reduced expression of ZO-1 in intestinal epithelial cells. In an in-vitro model, stimulation of human colon epithelial cells with peptidoglycan, but not with lipopolysaccharide, resulted in elevated secretion of pro-inflammatory cytokines, reflecting inflammatory activity of the intestinal epithelium.Taken together, PT and HS lead to increased permeability of the gut-blood barrier. Bacterial components may lead to production of inflammatory and chemotactic mediators by gut epithelial cells, underlining the role of the gut as an immunologically active organ.

Blood purification by nonselective hemoadsorption prevents death after traumatic brain injury and hemorrhagic shock in rats.

Patients who sustain traumatic brain injury (TBI) and concomitant hemorrhagic shock (HS) are at high risk of high-magnitude inflammation which can lead to poor outcomes and death. Blood purification by hemoadsorption (HA) offers an alternative intervention to reduce inflammation after injury. We tested the hypothesis that HA would reduce mortality in a rat model of TBI and HS. Male Sprague Dawley rats were subjected to a combined injury of a controlled cortical impact to their brain and pressure-controlled HS. Animals were subsequently instrumented with an extracorporeal blood circuit that passed through a cartridge for sham or experimental treatment. In experimental animals, the treatment cartridge was filled with proprietary beads (Cytosorbents, Monmouth Junction, NJ) that removed circulating molecules between 5 kDa and 60 kDa. Sham rats had equivalent circulation but no blood purification. Serial blood samples were analyzed with multiplex technology to quantify changes in a trauma-relevant panel of immunologic mediators. The primary outcome was survival to 96 hours postinjury. Hemoadsorption improved survival from 47% in sham-treated rats to 86% in HA-treated rats. There were no treatment-related changes in histologic appearance. Hemoadsorption affected biomarker concentrations both during the treatment and over the ensuing 4 days after injury. Distinct changes in biomarker concentrations were also measured in survivor and nonsurvivor rats from the entire cohort of rats indicating biomarker patterns associated with survival and death after injury. Blood purification by nonselective HA is an effective intervention to prevent death in a combined TBI/HS rat model. Hemoadsorption changed circulating concentrations of multiple inmmunologically active mediators during the treatment time frame and after treatment. Hemoadsorption has been safely implemented in human patients with sepsis and may be a treatment option after injury.

Hemorrhagic shock alters fracture callus composition and activates the IL6 and RANKL/OPG pathway in mice.

Fracture and hemorrhagic shock often lead to impaired fracture healing. To elucidate underlying pathogenesis, this study aimed to analyze histological properties during fracture healing after hemorrhagic shock and involved signaling pathways in mice. Male C57BL/6NCrl mice were assigned into five groups. Control group underwent no interventions. Sham group had a catheter and external fixator but neither blood loss nor osteotomy. Trauma-hemorrhage (TH) group received a pressure-controlled hemorrhagic shock; osteotomy (Fx) group, an osteotomy and fixator; and combined trauma (THFx) group, both hemorrhagic shock and externally fixed osteotomy. After 1, 2, 3, and 4 weeks, the animals were killed. Undecalcified bones were analyzed histologically and signaling pathways relevant for fracture healing by polymerase chain reaction and Western blot. Statistical significance was set at 0.05 or less. Comparisons were performed using the Mann-Whitney U or Kruskal-Wallis test. In the THFx group, a decreased bone formation after 3 weeks, a reduction of both bone and cartilage after 2 weeks, and an enhanced activation of the RANKL/OPG and IL6 signaling pathway after 1 week were shown in comparison to Fx. Hemorrhagic shock has a retarding effect on fracture healing in the early phase of fracture healing and leads to activation of the IL6 and RANKL/OPG signaling pathways.

Prolonged severe hemorrhagic shock at a mean arterial pressure of 40 mmHg does not lead to brain damage in rats.

AimTo examine whether prolonged hemorrhagic shock (HS) at a mean arterial pressure (MAP) of 40 mmHg leads to brain damage.MethodsRats were anesthetized with sevoflurane. The HS model consisted of the following phases: I, pressure‐controlled HS at a MAP of 40 mmHg; II, fluid resuscitation to normalize blood pressure; III, observations with outcome evaluations in terms of survival, overall performance categories, and neurological deficit scores, as well as evaluation of apoptosis in the hippocampus at 96 h. Each group of six rats was randomized into 60 min (group 1) or 75 min (group 2) each of phases I and II. Three sham rats were anesthetized for 150 min, and then awakened during phase III.ResultsThe three sham rats as well as five and two of the six rats in groups 1 and 2 (P < 0.05), respectively, survived for up to 96 h. All survivors were functionally normal with overall performance category = 1 and neurological deficit score = 0 at 96 h. Apoptotic neurons were not found in the hippocampus.ConclusionsThe higher mortality in group 2 suggested a more profound effect of HS compared with group 1. However, prolonged HS for 60 or 75 min did not cause functional damage or apoptosis in the hippocampus. These findings suggest that prolonged HS at a MAP of 40 mmHg, as a level at which cerebral blood flow seems preserved by autoregulatory mechanisms, does not lead to brain damage.

Lactated Ringer' Solution may be Superior to Saline-Based 6% Hydroxyethyl Starch 130/0.4 for Early Resuscitation within 12 hours from Hemorrhagic Shock

ABSTRACTPurpose: To compare the effects of fluid resuscitation with lactated Ringer's solution (LR) and saline-based 6% hydroxyethyl starch 130/0.4 (HES) on the inflammatory response and oxidative stress in the small intestine as well as on bacterial translocation to the liver. Methods: Sprague–Dawley rats were subjected to blood pressure-controlled hemorrhagic shock and then resuscitated with LR or HES. At 1, 3, 6, 12, and 24 hr after resuscitation, liver tissues were collected to count the bacterial colonies, and small intestines were harvested to analyze the levels of inflammatory (TNF-α and HO-1) and oxidative stress (MPO) mediators as well as the intestinal injury by immunohistochemistry, colorimetry and hematoxylin & eosin staining, respectively. Results: The expression level of TNF-α in the LR group was stable from 1 to 6 hr but decreased at 12 hr and then abruptly increased at 24 hr. The expression level of TNF-α in the LR group was significantly lower than that in the HES group, especially during the first 12 hr post-fluid infusion. MPO activity decreased to its lowest level at 3 hr but increased from 6 to 12 hr, with no difference at 24 hr between the two groups. Although a decreasing tendency was observed from 6 hr, HO-1 expression levels remained higher in the LR group than in the HES group at 12 and 24 hr, particularly at 12 hr. During the initial 12 hr, the LR group exhibited significantly lower colony-forming units in the liver tissues than the HES group. Chiu's score in the intestine decreased regardless of which resuscitative fluids were used. Conclusions: During early resuscitation (within 12 hr), LR may be superior to HES in reducing intestinal injuries by suppressing inflammatory and oxidative mediators.

Severe Hemorrhagic Shock Leads to a Delayed Fracture Healing and Decreased Bone Callus Strength in a Mouse Model.

Multiple trauma is frequently associated with hemorrhagic shock and fractures of the extremities. Clinically, the rate of impaired fracture healing (delayed healing and nonunion) seems to be increased in patients with multiple injuries compared with patients with isolated fractures. As the underlying pathogenesis remains poorly understood, we aimed to analyze the biomechanical properties during fracture healing in a murine model. The aim of this study was to determine whether fracture healing after severe hemorrhagic shock results in (1) delayed bridging as determined by macroscopic and radiographic assessment, (2) altered conditions of callus components as determined by µCT, and (3) decreased maximum bending moment measured by a three-point-bending test compared with ordinary fracture healing. Male C57BL/6NCrl mice were randomly assigned to five groups and four different times (five to 10 mice per group and time). Only the right femur from each mouse was used for analysis: the trauma hemorrhage (TH) group received a pressure-controlled hemorrhagic shock via catheter; the osteotomy (Fx) group underwent osteotomy and implantation of an external fixator on the right femur; the combined trauma (THFx) group received hemorrhagic shock and an external fixator with osteotomy; the sham group underwent implantation of a catheter and external fixator but had no blood loss or osteotomy, and the control group underwent no interventions. After 2, 3, 4, or 6 weeks, five to 10 animals of each group were sacrificed. Bones were analyzed macroscopically and via radiographs, µCT, and three-point-bending test. Statistical significance was set at a probability less than 0.05. Comparisons were performed using the Mann-Whitney U or the Kruskal-Wallis test. In the Fx group, the osteotomy gap was stable and bridged after 2 weeks in contrast to some bones in the THFx group where stable bridging did not occur. No difference was observed between the groups. µCT analysis showed reduced density of bone including callus (THFx: 1.17 g/cm3; interquartile range [IQR], 0.04 g/cm3; Fx: 1.22 g/cm3; IQR, 0.04 g/cm3; p=0.002; difference of medians [DM], -0.048; 95% CI, -0.073 to -0.029) and increased share of callus per volume of bone mass (%) after 2 weeks in the THFx group compared with the Fx group (THFx: 44.16%; IQR, 8.66%; Fx: 36.73%; IQR, 4.39%; p=0.015; DM, 7.634; 95% CI, 2.018-10.577). The three-point-bending test established a decreased maximum bending moment in the THFx group compared with the Fx group 2 weeks after surgery (THFx: 7.10 Nmm; IQR, 11.25 Nmm; Fx: 11.25 Nmm; IQR, 5.70 Nmm; p=0.026; DM, -5.043; 95% CI, -10.867 to -0.74). No differences were observed between the THFx and Fx groups after more than 2 weeks. In this in vivo mouse fracture model, we conclude that hemorrhagic shock retards fracture healing during the early phase of the facture healing process. A severe hemorrhagic shock in patients could result in initial delayed fracture healing and needs special attention. We plan to conduct a prospective, observational clinical research study to analyze if delayed fracture healing occurs in patients after severe blood loss.

Abstract 12591: Preservation of Cardiac Structure and Function Compared to Brain in a Rat Hemorrhagic Shock Model

Background: Hemorrhagic shock (HS) and resuscitation were hypothesized to induce myocardial injury and/or stunned myocardium. In this study we assessed cardiac function during HS and resuscitation in rats. Methods: Pressure-controlled HS was induced by blood shedding (withdrawal at 0.5 ml/min) in rats (n=5). Mean arterial blood pressure (MBP) was maintained at 29 ± 1 mmHg for 2 hours, followed by resuscitation with the shed blood. At 3 hours after resuscitation, saline was infused at 0.5 ml/min (2.5 ml) to assess cardiac and vascular reactivity. Cardiac function was continuously monitored by transthoracic echocardiogram during the procedure. Brain, heart, liver and kidney were sliced and stained with triphenyltetrazolium chloride for necrosis assessment. Results: No rat died during the procedure. Heart rate, MBP, diastolic and systolic left ventricular (LV) diameter, and LV fractional shortening (LVFS) were reported at 6 time points in the table: 1) baseline prior to HS; 2) beginning of HS; 3) end of the HS; 4) post-resuscitation peak MBP; 5) end of 3 hours of post-resuscitation; 6) 5 minutes after saline infusion at 0.5 ml/min. Heart rate significantly decreased during blood shedding (p &lt; 0.05), then significantly increased during HS (p &lt; 0.05). MBP significantly fell during HS (P &lt; 0.05) and then recovered to baseline level after reinfusion of shed blood; it significantly dropped (compared to baseline) at 3 hours after resuscitation (p &lt; 0.05). Saline infusion rapidly recovered MBP, LV diastolic diameter and LVFS to baseline level, which suggested that cardiac and vascular reactivity to fluid resuscitation were preserved during HS and post-resuscitation. LVFS remained preserved throughout the study; shock did not cause stunned myocardium. There were infarcts in 5 brains; no necrosis was detected in hearts, kidneys or livers. Conclusions: Cardiac function was preserved during HS and resuscitation with no evidence of stunning or infarction, while strokes were common.

Long-Term Effects of Induced Hypothermia on Local and Systemic Inflammation - Results from a Porcine Long-Term Trauma Model.

BackgroundHypothermia has been discussed as playing a role in improving the early phase of systemic inflammation. However, information on the impact of hypothermia on the local inflammatory response is sparse. We therefore investigated the kinetics of local and systemic inflammation in the late posttraumatic phase after induction of hypothermia in an established porcine long-term model of combined trauma.Materials & MethodsMale pigs (35 ± 5kg) were mechanically ventilated and monitored over the study period of 48 h. Combined trauma included tibia fracture, lung contusion, liver laceration and pressure-controlled hemorrhagic shock (MAP < 30 ± 5 mmHg for 90 min). After resuscitation, hypothermia (33°C) was induced for a period of 12 h (HT-T group) with subsequent re-warming over a period of 10 h. The NT-T group was kept normothermic. Systemic and local (fracture hematoma) cytokine levels (IL-6, -8, -10) and alarmins (HMGB1, HSP70) were measured via ELISA.ResultsSevere signs of shock as well as systemic and local increases of pro-inflammatory mediators were observed in both trauma groups. In general the local increase of pro- and anti-inflammatory mediator levels was significantly higher and prolonged compared to systemic concentrations. Induction of hypothermia resulted in a significantly prolonged elevation of both systemic and local HMGB1 levels at 48 h compared to the NT-T group. Correspondingly, local IL-6 levels demonstrated a significantly prolonged increase in the HT-T group at 48 h.ConclusionA prolonged inflammatory response might reduce the well-described protective effects on organ and immune function observed in the early phase after hypothermia induction. Furthermore, local immune response also seems to be affected. Future studies should aim to investigate the use of therapeutic hypothermia at different degrees and duration of application.

The effect of cyclosporine A in hemorrhagic shock model of rats.

The inhibition of mitochondrial permeability transition pore opening during ischemia-reperfusion can ameliorate injuries. This study aimed to investigate the effects of cyclosporine A (CsA) in rats after hemorrhagic shock. Male Sprague-Dawley rats were subjected to pressure-controlled hemorrhagic shock (mean arterial pressure, 38 ± 1 mm Hg) for 90 minutes. After the hemorrhagic shock period, rats were randomly allocated to one of three groups as follows: a control group, a CsA10 group, or a CsA50 group. CsA for the treatment groups (10 mg/kg for the CsA10 group and 50 mg/kg for the CsA50 group) or normal saline for the control group was administered via tail vein for 10 minutes, and shed blood was transfused for 15 minutes. For the survival study, animals were observed for up to 9 hours, and their survival time was recorded until death. Separate experiments were performed to examine the effect of CsA on inflammatory responses and liver injury. Rats were sacrificed at 210 minutes after the shock period, and blood and liver tissues were harvested. Survival times were shown to be significantly longer in the CsA-treated groups (i.e., the CsA10 and CsA50 groups) than in the control group. Plasma interleukin-6 and thiobarbituric acid-reactive substances were significantly lower in the CsA50 group than in the control group and phosphorylation of Akt, GSK-3β, and Bad were significantly increased in the CsA-treated groups compared with the control group. Expressions of Bcl-2, cleaved caspase 3, and cytoplasmic cytochrome C were significantly decreased in the CsA-treated groups compared with the control group. Although histologic liver injury was not significantly different among the groups, ultrastructural changes of mitochondria were more prominent in the control group than in the CsA-treated groups. CsA increased survival time, decreased proinflammatory cytokine and lipid peroxidation, and augmented Akt survival pathways in rats subjected to pressure-controlled hemorrhagic shock.

Extracorporeal membrane oxygenation combined with ultrafiltration technique for treatment of kidney injury after severe hemorrhagic shock in rabbits

Objective To investigate the effect of extracorporeal membrane oxygenation (ECMO) combined with ultrafiltration in treatment of kidney injury induced by serious hemorrhagic shock in rabbits.Methods Models of pressure-controlled hemorrhagic shock was developed in 24 New Zealand white rabbits which were divided into unresuscitation group (n =8),ECMO combined with ultrafiltration group (combined resuscitation group,n =8),and fluid resuscitation group (n =8) according to the random number table.Heart rate was monitored via electrocardiograph and arterial pressure via fermoral artery catheter.Blood samples were collected pre-and post-shock and after resuscitation to measure levels of lactic acid,serum creatinine,IL-6,and TNF-α.Kidney samples were collected for measurement of histopathological changes via HE staining,expression of heat shock protein 70 (HSP70) via immunohistochemical staining.Results Arterial pressure was (53.1 ± 11.4) mmHg in combined resuscitation group,higher than (41.3 ± 11.1) mmHg in fluid resuscitation group and (25.9 ± 10.5) mmHg in unresuscitation group (F =41.425,P ＜ 0.05).Hemorrhagic shock induced significant up-regulation of lactic acid,serum creatinine,IL-6,and TNF-α(P ＜ 0.05),but all were lowered after resuscitation,especially in combined resuscitation group (P ＜ 0.05).HE staining showed the degree of kidney tissue necrosis and inflammatory cytokine infiltration in combined resuscitation group alleviated notably compared with fluid resuscitation group.Median and interquartile values of HSP70 were 17 828.960 0 (15 779.865 0-21 751.980 0) in unresuscitation group,2 714.270 0 (1 339.215 0-7 616.950 0) in fluid resuscitation group,and 262.930 0 (198.820 0-538.195 0) in combine resuscitation group,with statistical differences among groups(P ＜ 0.05).Conclusion ECMO combined with ultrafiltration is superior to conventional fluid resuscitation in improving hypoxia tissue injury and inflammatory reaction after hemorrhagic shock and is beneficial to attenuating kidney injury. Key words: Shock, hemorrhagic ; Extracorporeal membrane oxygenation ; Ultrafiltration

Superimposed traumatic brain injury modulates vasomotor responses in third-order vessels after hemorrhagic shock

BackgroundTraumatic brain injury (TBI) and hemorrhagic shock (HS) are the leading causes of death in trauma. Recent studies suggest that TBI may influence physiological responses to acute blood loss. This study was designed to assess to what extent superimposed TBI may modulate physiologic vasomotor responses in third-order blood vessels in the context of HS.MethodsWe have combined two established experimental models of pressure-controlled hemorrhagic shock (HS; MAP 50 mmHg/60 min) and TBI (lateral fluid percussion (LFP)) to assess vasomotor responses and microcirculatory changes in third-order vessels by intravital microscopy in a spinotrapezius muscle preparation. 23 male Sprague–Dawley rats (260–320 g) were randomly assigned to experimental groups: i) Sham, ii) HS, iii) TBI + HS, subjected to impact or sham operation, and assessed.ResultsHS led to a significant decrease in arteriolar diameters by 20% to baseline (p < 0.01). In TBI + HS this vasoconstriction was less pronounced (5%, non-significant). At completed and at 60 minutes of resuscitation arteriolar diameters had recovered to pre-injury baseline values. Assessment of venular diameters revealed similar results. Arteriolar and venular RBC velocity and blood flow decreased sharply to < 20% of baseline in HS and TBI + HS (p < 0.01). Immediately after and at 60 minutes of resuscitation, an overshoot in arterial RBC velocity (140% of baseline) and blood flow (134.2%) was observed in TBI + HS.ConclusionSuperimposed TBI modulated arteriolar and venular responses to HS in third-order vessels in a spinotrapezius muscle preparation. Further research is necessary to precisely define the role of TBI on the microcirculation in tissues vulnerable to HS.

The pulse pressure/heart rate ratio as a marker of stroke volume changes during hemorrhagic shock and resuscitation in anesthetized swine

Emergency physicians and anesthesiologists need accurate estimates of stroke volume when massive unexpected hemorrhage occurs. Using an animal model of hemorrhagic shock under general anesthesia, we hypothesized that the pulse pressure-heart rate ratio (PP/HR) would be an accurate marker of stroke volume changes during hemorrhage and resuscitation. In 16 swine under bispectral index-controlled, intravenous propofol-remifentanil anesthesia, pressure-controlled hemorrhagic shock was induced to achieve 30 mm Hg of mean arterial pressure, after which treatment was randomized to fluid (HES, n = 4), norepinephrine (NE, n = 4), both (HES + NE, n = 4), or neither (control, n = 4). Pulmonary artery thermodilution continuous cardiac output, stroke volume, and central arterial pressures were recorded at baseline (T0), after 30 minutes (T30) and 60 (T60) minutes of hemorrhage, during treatment (T90 and T120) and after blood retransfusion (T180). At T60, blood withdrawal was 995 (301) mL (38 [8] mL/kg), resulting in a 70% decrease in stroke volume and a 3.3-fold decrease in PP/HR (each p < 0.01). When stroke volume data pointed at T0, T30 and T60 were plotted against the various hemodynamic variables under study, the PP/HR ratio exhibited the strongest relationship to stroke volume (r = 0.72). The area under the receiver operating characteristic curve set to detect a 15% stroke volume decrease was larger for PP/HR (0.95 [0.94-0.97]) than for mean arterial pressure (0.91 [0.89-0.93]) (p < 0.013). During resuscitation in the HES and NE groups, correlation coefficients were significantly higher between stroke volume and PP/HR (0.75 [0.63-0.84] and 0.79 [0.67-0.86]) than between stroke volume and mean arterial pressure (0.52 [0.32-0.67], p = 0.042, and 0.49 [0.28-0.65], p = 0.0018, respectively). The PP/HR ratio was strongly related to stroke volume during hemorrhagic shock and resuscitation in anesthetized swine.

The effect of hydrogen on hemorrhagic shock induced acute lung injury in rats

To investigate the effect of hydrogen inhalation on acute lung injury after hemorrhagic shock in rats. Twenty-four adult male Sprague-Dawley (SD) rats were equally randomized into three groups: sham operation group, model group and hydrogen-treatment group. Pressure-controlled hemorrhagic shock and resuscitation model was reproduced by blood-letting for 1 hour followed by fluid replacement for 2 hours. The rats in model group received a mixture of 50% oxygen-50% nitrogen during the process. The rats in hydrogen-treatment group received inhalation of a mixture of 2% hydrogen-48% nitrogen-50% oxygen 10 minutes before fluid replacement till the end of resuscitation. The arterial blood samples were collected for the measurement of arterial partial pressure of oxygen (PaO₂) before exsanguination, 1 hour after shock, 1 hour and 2 hours after fluid replacement. Blood and lung tissues were collected at the end of experiment, and tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in plasma, lung wet/dry weight ratio (W/D), malondialdehyde (MDA) content, superoxide dismutase (SOD) and myeloperoxidase (MPO) activity in the lung tissue were determined. The lung tissue was subjected to pathological examination. At the end of fluid replacement, compared with model group, hydrogen could significantly reduce pulmonary edema (lung W/D ratio: 4.72 ± 0.12 vs. 4.94 ± 0.14, P<0.05), inhibit oxidative stress (MDA: 0.55 ± 0.09 nmol/mg vs. 0.72 ± 0.08 nmol/mg, P<0.05), enhance antioxidant activity (SOD activity: 79.53 ± 14.33 U/mg vs. 59.55 ± 9.07 U/mg, P<0.05), reduce the release of pro-inflammatory cytokines (TNF-α: 55.58 ± 10.06 ng/L vs. 66.58 ± 5.17 ng/L; IL-6: 23.00 ± 2.77 ng/L vs. 27.09 ± 2.46 ng/L, both P<0.05) and inhibit neutrophil infiltration (MPO: 1.05 ± 0.18 U/g vs. 1.40 ± 0.14 U/g, P<0.05). It alleviated the damage to lung tissue, and then improved the lung function (PaO₂: 146.3 ± 22.1 mm Hg vs. 123.6 ± 16.0 mm Hg, P<0.05). Hydrogen treatment can alleviate acute lung injury as a result of hemorrhagic shock and resuscitation.