Publications by authors named "Naresh Ahuja"

5 Publications

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Impact of resuscitation strategies on the acetylation status of cardiac histones in a swine model of hemorrhage.

Resuscitation 2008 Feb 5;76(2):299-310. Epub 2007 Sep 5.

Trauma Research and Readiness Institute for Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.

Background: Chromatin remodeling through histone acetylation is a key control mechanism in gene transcription. We have shown previously that fluid resuscitation in rodents is coupled with highly structured post-translational modifications of cardiac histones. The current experiment was performed to validate this concept in a clinically relevant large animal model of hemorrhage and resuscitation, and to correlate the changes in histone acetylation with altered expression of immediate-early response genes.

Study Design: Yorkshire swine (n=49, 7/group, weight=40-58kg) were subjected to combined uncontrolled and controlled hemorrhage (40% of estimated blood volume) and randomly assigned to the following resuscitation groups: (1) 0.9% saline (NS), (2) racemic lactated Ringer's (dl-LR), (3) l-isomer lactated Ringer's (l-LR), (4) Ketone Ringer's (KR), (5) 6% hetastarch in saline (Hespan). KR contained an equimolar substitution of lactate with beta-hydroxybutyrate. No hemorrhage (NH) and no resuscitation (NR) groups were included as controls. Cardiac protein was used in Western blotting to analyze total protein acetylation and histone acetylation specifically. Lysine residue-specific acetylation of histone subunits H3 and H4 was further evaluated. In addition, Chromatin Immunoprecipitation (ChIP) technique was used to separate the DNA bound to acetylated histones (H3 and H4 subunits), followed by measurement of genes that are altered by hemorrhage/resuscitation, including immediate-early response genes (c-fos and c-myc), and heat shock protein (HSP) 70.

Results: The type of fluid used for resuscitation influenced the patterns of cardiac histone acetylation. Resuscitation with dl-LR and KR induced hyperacetylation on H3K9. KR resuscitation was also associated with increased acetylation on H3K14 and H4K5, and hypoacetylation on H3K18. The expression of genes was also fluid specific, with the largest number of changes following KR resuscitation (increased c-fos and c-myc, HSP 70 linked with H3; and increased c-myc linked with H4). Among the histone subunits studied, altered H3 acetylations were associated with the majority of changes in immediate-early gene expression.

Conclusions: Acetylation status of cardiac histones, affected by hemorrhage, is further modulated by resuscitation producing a fluid-specific code that is preserved in different species. Resuscitation with KR causes histone acetylation at the largest number of lysine sites (predominately H3 subunit), and has the most pronounced impact on the transcriptional regulation of selected (immediate-early response) genes.
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February 2008

Profound hypothermic cardiopulmonary bypass facilitates survival without a high complication rate in a swine model of complex vascular, splenic, and colon injuries.

J Am Coll Surg 2007 Apr 2;204(4):642-53. Epub 2007 Mar 2.

Division of Trauma, Emergency Surgery and Surgical Critical Care, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114, USA.

Background: Induction of a profound hypothermia for emergency preservation and resuscitation in severe hemorrhagic shock can improve survival from lethal injuries, but the impact of hypothermia on bleeding and infectious complications has not been completely determined.

Study Design: Uncontrolled hemorrhage was induced in 26 swine (95 to 135 lbs) by creating an iliac artery and vein injury, and 30 minutes later, by lacerating the descending thoracic aorta. Through a left thoracotomy approach, profound total body hypothermia (10 degrees C) was induced (2 degrees C/min) by infusing cold organ preservation solution into the aorta. The experimental groups were: vascular injuries alone (group 1, n=10), vascular and colon injuries (group 2, n=8), and vascular, colon, and splenic injuries (group 3, n=8). All injuries were repaired during 60 minutes of low-flow cardiopulmonary bypass (CPB) with hemodilution and profound hypothermia; then the animals were slowly rewarmed (0.5 degrees C/min) back to normothermia. Survivors were monitored for 6 weeks for postoperative bleeding, neurologic deficits, cognitive function (learning new skills), organ dysfunction, and septic complications.

Results: Six-week survival rates were 90% in group 1, 87.5% in group 2, and 75% in group 3 (p > 0.05). One animal in each group died from acute cardiac failure during the early postoperative phase. Splenic salvage was possible in all animals, and none required complete splenectomy for hemorrhage control. All surviving animals were neurologically intact, displayed normal learning capacity, and had no longterm organ dysfunction. None of the animals had postoperative hemorrhage or experienced septic complications. One animal in group 3 died on the ninth postoperative day because of bowel obstruction (volvulus).

Conclusions: Induction of profound hypothermia can preserve the viability of key organs during repair of lethal injuries. This strategy can be used even in the presence of solid organ and bowel injuries to improve survival, without any considerable increase in postoperative complication rates.
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April 2007

Testing of modified zeolite hemostatic dressings in a large animal model of lethal groin injury.

J Trauma 2006 Dec;61(6):1312-20

Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.

Background: We have previously identified a granular zeolite hemostat (ZH) as an effective agent for control of severe bleeding, and it is currently being used by the US troops in the battlefield. ZH causes an exothermic reaction on application, which theoretically can be decreased by altering its chemical composition or changing its physical properties. However, the effect of these alterations on the hemostatic efficacy is unknown. We tested modified zeolites and a chitosan based dressing against controls in a swine model of battlefield injury.

Methods: A complex groin injury was created in 60 swine (40-55 kg). This included semi-transection of the proximal thigh (level of inguinal ligament), and complete division of the femoral artery and vein. After 3 minutes, the animals were assigned to (1) no dressing (ND), (2) standard dressing (SD), (3-5) SD + chemically modified ZHs, where calcium was substituted with sodium (Na), barium (Ba), or silver (Ag), respectively, (6) SD + physically modified ZH, where "beads" were packaged in a fabric bag, (7) SD + chitosan based dressing (CD). Resuscitation was started 15 minutes after application of dressing (500 mL of 6% hetastarch over 30 minutes). Survival for 180 minutes was the primary endpoint for this study. In addition, blood loss, wound temperatures, and histologic tissue damage were recorded.

Results: Mortality in the group that was treated with the application of bagged ZH was 10% versus 100% in the no dressing group and 50% in the SD group (p < 0.05 vs. ND and SD groups). The Na ZH group had a mortality rate of 43%, whereas application of Ba and Ag substituted zeolites, and CD were associated with a mortality rate of 25%. Ionic substitution of zeolite decreased the in vivo temperature peak by 5 to 10 degrees C. No histologic evidence of tissue necrosis was noted in this experiment.

Conclusions: The use of zeolite hemostat can control hemorrhage and dramatically reduce mortality from a lethal groin wound. Modifications of zeolite hemostat can decrease the exothermic reaction and attenuate tissue damage.
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December 2006

Hepatic and pulmonary apoptosis after hemorrhagic shock in swine can be reduced through modifications of conventional Ringer's solution.

J Trauma 2006 Jan;60(1):52-63

Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.

Background: Cytotoxic properties of racemic (D-,L-isomers) lactated Ringer's solution detected in vitro and in small animal experiments, have not been confirmed in large animal models. Our hypothesis was that in a clinically relevant large animal model of hemorrhage, resuscitation with racemic lactated Ringer's solution would induce cellular apoptosis, which can be attenuated by elimination of d-lactate.

Methods: Yorkshire swine (n = 49, weight 40-58 kg) were subjected to uncontrolled (iliac arterial and venous injuries) and controlled hemorrhage, totaling 40% of estimated blood volume. They were randomized (n = 7/group) to control groups, which consisted of (1) no hemorrhage (NH), (2) no resuscitation (NR), or resuscitation groups, which consisted of (3) 0.9% saline (NS), (4) racemic lactated Ringer's (DL-LR), (5) L-isomer lactated Ringer's (L-LR), (6) Ketone Ringer's (KR), (7) 6% hetastarch in 0.9% saline (Hespan). KR was identical to LR except for equimolar substitution of lactate with beta-hydroxybutyrate. Resuscitation was performed in three phases, simulating (1) prehospital, (2) operative, (3) postoperative/recovery periods. Arterial blood gasses, circulating cytokines (TNF-alpha, IL-1, -6, -10), and markers of organ injury were serially measured. Metabolic activity of brain, and liver, was measured with microdialysis. Four hours postinjury, organs were harvested for Western blotting, ELISA, TUNEL assay, and immunohistochemistry.

Results: All resuscitation strategies restored blood pressure, but clearance of lactic acidosis was impeded following DL-LR resuscitation. Metabolic activity decreased during shock and improved with resuscitation, without any significant inter-group differences. Levels of cytokines in circulation were similar, but tissue levels of TNF in liver and lung increased six- and threefolds (p < 0.05) in NR group. In liver, all resuscitation strategies significantly decreased TNF levels compared with the NR group, but in the lung resuscitation with lactated Ringer (DL and L isomers) failed to decrease tissue TNF levels. DL-LR resuscitation also increased apoptosis (p < 0.05) in liver and lung, which was not seen after resuscitation with other solutions.

Conclusions: In this large animal model of hemorrhagic shock, resuscitation with conventional (racemic) LR solution increased apoptotic cell death in liver and lung. This effect can be prevented by simple elimination of D-lactate from the Ringer's solution.
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January 2006

Profound hypothermia protects neurons and astrocytes, and preserves cognitive functions in a Swine model of lethal hemorrhage.

J Surg Res 2005 Jun;126(2):172-81

Trauma Research and Readiness Institute for Surgery, Bethesda, Maryland, USA.

Background: Lethal injuries can be repaired under asanguineous hypothermic arrest (suspended animation) with excellent survival. This experiment was designed to test the impact of this strategy on neuronal and astroglial damage in a swine model of lethal hemorrhage. Furthermore, our goal was to correlate the histological changes in the brain with neurological outcome, and the levels of circulating brain specific markers.

Materials And Methods: Uncontrolled hemorrhage was induced in 32 female swine (80-120 lbs) by creating an iliac artery and vein injury, followed 30 min later by laceration of the thoracic aorta. Through a thoracotomy approach, organ preservation fluid was infused into the aorta using a roller pump. Experimental groups included normothermic controls (no cooling, NC), and groups where hypothermia was induced at three different rates: 0.5 degrees C/min (slow, SC), 1 degrees C/min (medium, MC), or 2 degrees C/min (fast, FC). Profound hypothermia (core temperature of 10 degrees C) was maintained for 60 min for repair of vascular injuries, after which the animals were re-warmed (0.5 degrees C/min) and resuscitated on cardiopulmonary bypass (CPB). Circulating levels of neuron specific enolase (NSE) and S-100beta were serially measured as markers of damage to neurons and astrocytes, respectively. Light microscopy and quantitative immunohistochemical techniques were used to evaluate hippocampal CA1 area and caudate putamen for neuronal injury and astrogliosis (astrocyte hyperplasia/hypertrophy). Surviving animals were observed for 6 weeks and neurological status was documented on an objective scale, and cognitive functions were evaluated using a technique based upon the concept of operant conditioning.

Results: Normothermic arrest resulted in clinical brain death in all of the animals. None of the surviving hypothermic animals displayed any neurological deficits or cognitive impairment. On histological examination, normothermic animals were found to have ischemic changes in the neurons and astrocytes (hypertrophy). In contrast, all of the hypothermic animals had histologically normal brains. The circulating levels of brain specific proteins did not correlate with the degree of brain damage. The changes in NSE levels were not statistically significant, whereas S-100beta increased in the circulation after CPB, largely independent of the temperature modulation.

Conclusions: Profound hypothermia can preserve viability of neurons and astrocytes during prolonged periods of cerebral hypoxia. This approach is associated with excellent cognitive and neurological outcome following severe shock. Circulating markers of central nervous system injury did not correlate with the actual degree of brain damage in this model.
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June 2005