Hasan B. Alam

Application of a zeolite hemostatic agent achieves 100% survival in a lethal model of complex groin injury in swine

BACKGROUNDnTechniques for better hemorrhage control after injury could change outcome. We have previously shown that a zeolite mineral hemostatic agent (ZH) can control aggressive bleeding through adsorption of water, which is an exothermic process. Increasing the residual moisture content (RM) of ZH can theoretically decrease heat generation, but its effect on the hemostatic properties is unknown. We tested ZH with increasing RM against controls and other hemostatic agents in a swine model of battlefield injury.nnnMETHODSnA complex groin injury was created in 72 swine (37 +/- 0.8 kg). This included semitransection of the proximal thigh and complete division of the femoral artery and vein. After 3 minutes, the animals were randomized to 1 of 10 groups: group 1, no dressing (ND); group 2, standard dressing (SD); group 3, SD + 3.5 oz ZH with 1% RM (1% ZH); group 4, SD + 3.5 oz ZH with 4% RM (4% ZH); group 5, SD + 2 oz ZH with 1% RM (1% ZH 2oz); group 6, SD + 3.5 oz ZH with 8% RM (8% ZH); group 7, SD + chitosan-based hemostat, HemCon (HC); group 8, SD + 3.5 oz nonzeolite mineral hemostat, Quick Relief (NZH); group 9, SD + bovine clotting factors-based hemostat, Fast Act (FA); and group 10, SD + 30 g of starch-based hemostat, TraumaDex (TDex). Resuscitation (500 mL of Hespan over 30 minutes) was started 15 minutes after injury and hemodynamic monitoring was performed for 180 minutes. Primary endpoints were survival for 180 minutes and blood loss. In addition, maximum wound temperatures were recorded, and histologic damage to artery, vein, nerve, and muscle was documented.nnnRESULTSnUse of 1% ZH decreased blood loss and reduced mortality to 0% (p < 0.05). Increasing the RM adversely affected efficacy without any significant decrease in wound temperatures. Minimal histologic tissue damage was seen with ZH independent of the percentage of RM.nnnCONCLUSIONnThe use of zeolite hemostatic agent (1% residual moisture, 3.5 oz) can control hemorrhage and dramatically reduce mortality from a lethal groin wound.

Hemorrhage control in the battlefield: Role of new hemostatic agents

Uncontrolled hemorrhage is the leading cause of preventable combat-related deaths. The vast majority of these deaths occur in the field before the injured can be transported to a treatment facility. Early control of hemorrhage remains the most effective strategy for treating combat casualties. A number of hemostatic agents have recently been deployed to the warfront that can be used to arrest bleeding before surgical control of the source. The purpose of this article is to summarize the background information regarding these hemostatic agents, indications and rationale for their use, and characteristics of these products that may impact effectiveness.

Comparative analysis of hemostatic agents in a swine model of lethal groin injury.

BACKGROUNDnTechniques for better hemorrhage control after injury could change outcome. A large-animal model of lethal, uncontrolled hemorrhage was developed to test whether the use of various hemostatic agents would decrease bleeding and improve early survival.nnnMETHODSnA complex groin injury was created in 30 Yorkshire swine (42-55 kg) to produce uncontrolled hemorrhage. This injury included semitransection of the proximal thigh and complete division of the femoral artery and vein. After 5 minutes, the animals were randomized to (n = 6 animals per group) no dressing (ND), standard dressing (SD), SD and Rapid Deployment Hemostat (RDH) bandage, SD and QuikClot hemostatic agent (QC), or SD and TraumaDEX (TDEX). Limited volume 0.9% saline (1,000 mL over 30 minutes) resuscitation was started 30 minutes after injury. We measured blood loss, early mortality (180 minutes), and physiologic markers of hemorrhagic shock (e.g., cardiac output, blood pressure, hemoglobin, metabolic acidosis).nnnRESULTSnApplication of wound dressing decreased mortality in all groups compared with the ND group (83% mortality). However, this difference was significant (p < 0.05) only for the QuikClot hemostatic agent (0% mortality). Before the application of dressing (first 5 minutes), there were no differences in blood loss between the groups. After application of dressings, the QC group had the lowest blood loss (4.4 +/- 1.4 mL/kg).nnnCONCLUSIONnOf the hemostatic agents tested, QuikClot improved survival and decreased bleeding in a swine model of lethal vascular and soft tissue injury.

Searching for the optimal resuscitation method: Recommendations for the initial fluid resuscitation of combat casualties

Resuscitation can exacerbate cellular injury caused by hemorrhagic shock, and the type of fluid used for resuscitation may play an important role in this injury. Unlike some factors in the treatment of combat casualty, the method of resuscitation is under our control. The prevention of cellular injury through wiser resuscitation strategies would be more advantageous than attempting complex immunomodulation after the damage has already occurred. This article summarizes data from a number of studies to illustrate the differential effects of commonly used resuscitation fluids on cellular injury. Our findings show that resuscitation with hypotonic/isotonic crystalloids, including lactated Ringers (LR) solution, and artificial colloid solutions, elicit severe immune activation and an up-regulation of cellular injury markers. This effect is not seen with plasma, natural colloids (albumin), and fresh whole blood. Hypertonic fluids cause suppression of neutrophil activation and a milder increase in the expression of cell injury markers compared with isotonic fluids. The effect of various resuscitation fluids on core cellular functions such as gene regulation is also summarized in this article. Finally, because of the uniqueness of combat care, a set of new recommendations for initial fluid resuscitation of combat casualties is proposed.

Effects of Lactated Ringer's Solutions on Human Leukocytes

BACKGROUNDnThe standard lactated Ringers (LR) solution contains racemic lactate, an equal mixture of D(-)- and L(+)-isomers. The aim of this study was to investigate whether racemic LR solution (containing both isomers, dl-LR) differs from LR containing L-isomer only (L-LR).nnnMETHODSnBlood from 20 volunteers was incubated for 30 minutes with lactated Ringers solutions containing the DL- or L-form of lactate, Hanks balanced salt solution, normal saline, and ketone Ringers (lactate replaced with ketone bodies). Neutrophil oxidative burst was measured using flow cytometry. Gene expression of 23 genes associated with leukocyte function was determined with cDNA array technique. The arraying procedure was repeated four times to obtain four sets of data.nnnRESULTSnCompared with the L-LR and ketone Ringers, DL-LR causes an increased production of reactive oxygen species by neutrophils and affects expression of leukocyte genes known to be involved in inflammation, cell migration, and apoptosis.nnnCONCLUSIONnLactated Ringers solution in commonly used formulation (racemic mixture, DL-LR) influences neutrophil function and leukocyte gene expression.

The Rate of Induction of Hypothermic Arrest Determines the Outcome in a Swine Model of Lethal Hemorrhage

BACKGROUNDnLethal injuries can be surgically repaired under asanguineous hypothermic condition (suspended animation) with excellent outcome. However, the optimal rate for the induction of hypothermic metabolic arrest following uncontrolled lethal hemorrhage (ULH) is unknown.nnnMETHODSnULH was induced in 32 female swine (80-120 lbs) by creating an iliac artery and vein injury, followed 30 minutes later by laceration of the descending thoracic aorta. Through a left thoracotomy approach, total body hypothermic hyperkalemic metabolic arrest was induced by infusing organ preservation fluids into the aorta. Experimental groups were: normothermic controls (no cooling, NC), or hypothermia induced at a rate of 0.5 degrees C/min (slow, SC), 1 degrees C/min (medium, MC), or 2 degrees C/min (fast, FC). Vascular injuries were repaired during the 60 minutes of profound (10 degrees C) hypothermic arrest. Hyperkalemia was reversed by hypokalemic fluid exchange, and blood was infused for resuscitation during the re-warming (0.5 degrees C/ minute) period. The survivors were monitored for 6 weeks.nnnRESULTSnThe 6 week survival rates were 0% (NC), 37.5% (SC), 62.5% (MC), and 87.5% (FC) respectively (p < 0.05 MC&FC versus NC). All of the surviving hypothermic arrest animals were neurologically intact and displayed no long term organ dysfunction.nnnCONCLUSIONnHypothermic metabolic arrest can be used to maintain viability of key organs during repair of lethal injuries. Survival is influenced by the rate of cooling with the best outcome following rapid induction of hypothermia.

Resuscitation-induced pulmonary apoptosis and intracellular adhesion molecule-1 expression in rats are attenuated by the use of Ketone Ringer's solution.

BACKGROUNDnResuscitation with Lactated Ringers solution after hemorrhagic shock in rats has been shown to cause early cellular injury in the lung. We hypothesized that the use of energy substrates, such as ketone bodies, in the resuscitation fluids would protect against this injury. As markers of cellular injury we measured the induction of apoptotic cell death and the expression of Intracellular Adhesion Molecule-1 (ICAM-1).nnnSTUDY DESIGNnMale Sprague Dawley rats (n = 35) under inhaled isoflurane anesthesia had placement of femoral arterial and venous catheters. A three-stage hemorrhage model was used for this experiment. There was an initial hemorrhage of 27 mL/kg for 10 minutes. During the next 75 minutes another 8 mL/kg of blood was withdrawn at a steady rate. The resuscitation fluids were then infused for 45 minutes during which the third continuous hemorrhage of 8 mL/kg was performed. The animals were randomized to five groups: 1) sham hemorrhage (n = 6); 2) sham resuscitation (n = 7); 3) Lactated Ringers resuscitation, three times the volume of shed blood (n = 8); 4) Ketone Ringers (containing 28 mEq/L of beta-hydroxybutyrate) resuscitation, three times the volume of shed blood (n = 7); and 5) plasma resuscitation, volume equal to shed blood (n = 7). The animals were sacrificed 1 hour after resuscitation and lungs were harvested. Western blot technique was used for the determination of proapoptotic protein (bax), antiapoptotic protein (bcl-2), apoptotic fragments of poly ADP-ribose polymerase, and ICAM-1. Sections of lung were also subjected to immunostaining using antibodies to bax and ICAM-1 proteins (reported as number of positive cells/mm2).nnnRESULTSnLactated Ringers resuscitation caused a significant increase in pulmonary apoptosis and ICAM-1 expression compared with the sham hemorrhage group. Animals resuscitated with Ketone Ringers solution and plasma did not show this injury pattern.nnnCONCLUSIONSnSubstitution of lactate with ketone bodies in the resuscitation fluid attenuates the expression of cellular injury markers in the lung.

cDNA array analysis of gene expression following hemorrhagic shock and resuscitation in rats

UNLABELLEDnThe aim of this study was to characterize gene expression following hemorrhagic shock and resuscitation with emphasis on the differences between various resuscitation strategies.nnnMETHODSnMale Sprague Dawley rats (n = 25; 5/group) were subjected to a three stage hemorrhage and resuscitated as follows: (1) sham hemorrhage; (2) sham resuscitation; (3) lactated Ringers solution (LR), 3:1 volume; (4) 7.5% hypertonic saline (HTS) 9.7 ml/kg; (5) plasma, 1:1 volume. Liver, spleen, lung and muscle were collected 3 h post resuscitation and cDNA array analysis was performed on the total RNA.nnnRESULTSnExpression of 1,176 genes was analyzed. Following resuscitation, 82 of the genes studied (7%) displayed an altered expression of at least 2-fold compared to the sham hemorrhage group. Depending on organ system under study and resuscitation conditions, expression of these 82 genes was down- or up-regulated, bringing the total number of expression alterations to 167. Largest number of organ-specific changes in gene expression was noted in liver (63/167), followed by lung (57), muscle (25), and spleen (22). Most of the resuscitation strategy specific changes were caused by plasma resuscitation (68/167), followed by LR (51), and HTS (48). In every organ studied, gene expression profile was dependent upon the fluid used for resuscitation.nnnCONCLUSIONnCellular response to hemorrhagic shock, even at the level of gene expression, is dependent on the resuscitation strategy. We have discovered altered expression of genes not previously implicated in the physiology of hemorrhagic shock and resuscitation. Gene array technology provides a rapid and efficient means of dissecting the complex genetic regulation of cellular response to shock.

Hypertonic saline: intraosseous infusion causes myonecrosis in a dehydrated swine model of uncontrolled hemorrhagic shock

BACKGROUNDnThe Institute of Medicine has recommended intraosseous (IO) infusion of 7.5% hypertonic saline (HTS) for combat casualties in shock. We tested the safety and efficacy of this recommendation in a long-term survival model of uncontrolled hemorrhagic shock using dehydrated swine.nnnMETHODSnFourteen dehydrated Yorkshire swine had placement of a 12G needle in the right anterior tibia under isoflurane anesthesia. Uncontrolled hemorrhage was induced via left iliac artery and vein injury. Animals were kept in shock for 2 hours and then resuscitated over 2 hours with 5 mL/kg of 7.5% HTS given either as 10 small boluses (group I, n = 4) or two large boluses (group II, n = 6) to compare the physiologic response and blood loss. Control animals (group III, n = 4) received an equal volume of 0.9% saline IO and additional intravenous saline to equalize the salt load in all groups.nnnRESULTSnThe three groups had similar physiologic responses, with no increase in blood loss following HTS resuscitation. However, between the second and fifth postresuscitation days, the 7.5% HTS resuscitated animals developed soft tissue necrosis or bone marrow necrosis of the right hind leg (group I, 100%; group II, 66.6%; group III, 0%).nnnCONCLUSIONnHTS resuscitation effectively restored hemodynamic stability in dehydrated swine without increased bleeding from an uncontrolled vascular injury. However, IO infusion of HTS in this model was associated with a very high rate of local complications. Further investigations should be undertaken before IO use of 7.5% HTS in humans.

Valproic acid prevents hemorrhage-associated lethality and affects the acetylation pattern of cardiac histones.

ABSTRACT Pharmacological inhibitors of histone deacetylases (HDAC) demonstrate cytoprotective effects both in vitro and in vivo. In this study, we investigated whether valproic acid (VPA), a known mood stabilizer and anticonvulsant with HDAC-inhibiting activity, improves survival following otherwise lethal hemorrhage in rats. We found that preinsult injection of VPA (300 mg/kg, twice) prolonged the survival of severely hypotensive animals up to 5 times. VPA treatment increased the acetylation of nonhistone and histone proteins in the rat heart. The pattern of modifications of individual histones revealed hyperacetylation of histones H2A, H3, and H4, indicating the presence of active genes. Expression of HSP70 and superoxide dismutase, implicated in the modulation of vitality, was increased by VPA. Our results reveal that VPA offers considerable protection in the hemorrhagic shock model and suggest a role for HDAC inhibition in mediating VPA actions.