31.06 High ratio FFP and platelet transfusion in nontrauma massive transfusion: too much of a good thing?

Posted on December 22, 2014

E. W. Etchill1, L. M. McDaniel1, S. P. Myers1, J. S. Raval2, A. B. Peitzman1, J. L. Sperry1, M. D. Neal1  1University Of Pittsburgh School Of Medicine,Department Of Surgery,Pittsburgh, PENNSYLVANIA, USA 2University Of North Carolina School Of Medicine,Division Of Transfusion Medicine,Chapel Hill, NORTH CAROLINA, USA

Introduction: Current resuscitation strategies in trauma focus on the delivery of fixed ratios of fresh frozen plasma (FFP) and platelets (PLT) along with packed red blood cells (PRBC) as part of massive transfusion protocols. Unfortunately, there is a paucity of evidence regarding the outcomes of massive transfusion protocols in the non-trauma setting where patients may exhibit a different coagulopathic profile. We hypothesized that the use of increased plasma and platelet to red blood cell ratios ( > 1:2) results in no significant difference in morbidity or mortality compared to lower plasma and platelet to red blood cell ratios ( ≤ 1:2). 

Methods: This was a 2-year retrospective single institutional analysis of massively transfused non-trauma patients. Pediatric and obstetric patients, patients on anticoagulants, and patients who died within 24 hours of transfusion were excluded. Ratios of fresh frozen plasma-packed red blood cell (PRBC) were calculated and divided in to a high ratio (FFP:PRBC >1:2) and a low-ratio (FFP:PRBC ≤1:2) group. Platelet ratios were calculated in the same manner. The primary outcomes of interest were 48-hour and 30-day mortality among patients receiving greater than 10 units of PRBC in a 24 hour period. Secondary outcomes included length of stay, ICU days, and ventilator free days. Logistical regression was utilized and a Cox regression survival analysis was performed after controlling for major co-morbidities, ASA and APACHE II scores.  
 

Results: Among 292 massively transfused non-trauma patients, cardiovascular surgery, GI bleeds, and intraoperative complications were the most common indications for massive transfusion. 48-hour mortality, post-transfusion hospital length of stay, ICU length of stay, and ventilator-free days were not significantly different between the high and low ratio FFP:PRBC groups. Interestingly, giving higher ratios of FFP (>1:2 FFP:PRBC) was significantly associated with decreased 30-day survival (66.6% vs. 82.2 %, p =0.0056).

30-day mortality, post-transfusion hospital length of stay, ICU length of stay, and ventilator-free days did not significantly differ between the high and low ratio PLT:PRBC groups. Using multivariate logistic regression analysis, neither FFP:PRBC nor PLT:PRBC ratios were predictors of 48-hour or 30-day mortality. 

Conclusion: Our study suggests that higher ratios of FFP and PLT to PRBC, which have been shown to provide a survival benefit in trauma populations, may be of limited benefit in the non-trauma, massively transfused population. Furthermore, a higher ratio of FFP may actually be associated with an increased mortality. Further prospective investigation into the appropriateness of balanced resuscitation in non-trauma populations is warranted. 

31.08 Old Blood and Complications in the Massively Transfused Trauma Patient: A Cautionary Tale

Posted on December 22, 2014

A. Lubitz1, K. Hollenbach2, E. Chan1, E. Dauer1, L. Sjoholm1, A. Pathak1, T. Santora1, A. Goldberg1, J. Rappold1  1Temple University School Of Medicine,Department Of Surgery,Philadelpha, PA, USA 2University Of California – San Diego,Skaggs School Of Pharmacy And Pharmeceutical Sciences,San Diego, CA, USA

Introduction:  The age of stored packed red blood cells (pRBC) and it's relationship to the development of complications in trauma patients requiring massive transfusions (≥ 10 units pRBC/24 hour) remain poorly understood. Laboratory studies have revealed evidence of the accumulation of storage lesions and decreased oxygen carrying capacity in pRBCs stored for greater than 14 days. The clinical significance of these findings on the development of both infectious and non-infectious complications remains unknown.

Methods: A retrospective cohort study of all massive transfusion (MTP) patients at an urban level 1 trauma center between 2008 and 2012 was conducted.   Data were abstracted from the trauma registry and corroborated with medical and blood bank records; included were standard demographic information, mechanism of injury, injury severity score (ISS), the amount and age of pRBCs transfused and patient outcomes.  Data were analyzed using STATA 13.1. Variables were created to examine the age of blood received in the MTP resuscitation by week and by < 14 days. Initial comparisons were made using t-tests and chi-square statistics, as appropriate.  Logistic regression was used to determine the odds of complication associated with percent of blood < 14 days of age.  Subsequent analyses were conducted independently for both infectious and non-infectious complications.

Results: There were a total of 133 MTP patients during the study period: 70 with at least one complication and 63 with no complications.  The mean age of patients with complications was slightly younger than those without complications (30.3±10.8 vs. 32.1±14.1).  Patients with complications had lower injury severity scores (ISS) than patients without complications (24.1±13.4 vs. 27.9±19.1).  Patients with complications were 3.3 times more likely to have 50% or more blood transfused that was  ≥ 14 days of age than patients with no complications (95% CI = 1.4, 8.3).  When the analysis was restricted to infectious complications, the effect of aged blood was greater (OR = 5.7; 95% CI = 1.7, 24.4). Neither age nor ISS changed these associations.

Conclusions: These data indicate that aged blood was associated with a significant increased risk of complications.  Further studies are warranted to determine what factors in blood ≥ 14 days of age may exist that puts massively transfused trauma patients at risk for the development of complications, particularly infections.

31.09 Mopeds: Not the Lesser Evil

Posted on December 22, 2014

M. Stawikowska1, B. L. Brewer1, B. L. Zarzaur1, J. Coleman1, D. V. Feliciano1, G. S. Rozycki1  1Indiana University School Of Medicine,Surgery,Indianapolis, IN, USA

Introduction:  Compared to motorcycles (MC), use of scooters and mopeds (MP) is largely unregulated as MP are perceived as safer than MC.  The objective of this retrospective study was to compare the demographics, injury patterns and injury severity of MP and MC crash victims.  The hypothesis was that the two groups will have similar injury patterns and severity of injuries.

Methods: This retrospective, observational study was performed by reviewing the trauma registry at an urban level 1 trauma center.  All patients admitted after a motorcycle crash (MCC) or moped crash (MPC) from 2010 – 2014 were included in the study.  Patient demographics, insurance and injury related factors were compared.

Results: 1194 patients met inclusion criteria (1031 MCC, 163 MPC).  Age and gender were similar between the two groups.  MP drivers were more likely to be non-white (p=0.001) than MC drivers, and were significantly less likely to wear a helmet (p<0.001) or to be insured (p<0.0001).  MP drivers were also more likely to be under the influence of alcohol (p<0.001) or drugs (p<0.001).  MP drivers were significantly more likely to sustain traumatic brain injury (p=0.002) than MC drivers, while patients in MCC were more likely to sustain spinal cord injuries (p=0.004).  Mean ISS and percent of patients with long bone fractures were similar between the two groups.

Conclusion: Despite being less regulated, victims of MPC are at least as likely, if not more likely, to suffer serious injury compared to victims of MCC.  Also, MP riders are more likely to ride helmetless and with drug or alcohol impairment.  While the use of MP may be perceived as safer than MC, stricter regulations governing this type of road vehicle should be pursued to prevent serious injury.

 

3.19 Hemoglobin Based Oxygen Carriers Exacerbate Hyperfibrinolysis Independent of Plasmin

Posted on December 22, 2014

A. P. Morton3, H. B. Moore3, E. Gonzalez3, G. Wiener3, P. Lawson2, M. Chapman3, C. Silliman3, E. Peltz1,3, A. Banerjee3, E. E. Moore2,3  1University Of Colorado Hospital,Surgery,Aurora, CO, USA 2Denver Health Medical Center,Surgery,Denver, CO, USA 3University Of Colorado School Of Medicine,Surgery,Aurora, CO, USA

Introduction: Hyperfibrinolysis plays an integral role in the genesis of trauma induced coagulopathy (TIC). Recent data demonstrates that red blood cell (RBC) lysis promotes fibrinolysis; however, the mechanism is unclear.  Hemoglobin-based oxygen carriers (HBOC) have been developed for resuscitation and have been associated with coagulopathy.  We hypothesize that replacement of whole blood (WB) using an HBOC results in a coagulopathy due to the presence of free hemoglobin.

Methods: Whole blood was sampled from healthy donors (n=6).  The clotting profile of each citrated sample was evaluated using native thromboelastography (TEG).  Serial titrations were performed using both HBOC (Polyheme) and normal saline (NS) (5%, 25%, and 50%) and evaluated both with and without a 75 ng/microliter tissue-plasminogen activator (tPA) challenge.  Tranexamic acid (TXA) was added to inhibit plasmin dependent fibrinolysis.  Fibrinolysis was measured and recorded as LY30, the percentage of clot lysis at 30 minutes after maximal clot strength.  Statistics were calculated using SPSS software.  Dilution of WB with NS or HBOC was correlated using LY30 via Spearman Rho coefficients.  Groups were also compared using a Friedman test and post-hoc analysis with a Bonferroni adjustment.

Results: TPA-provoked fibrinolysis was enhanced by both HBOC (median LY30 at 5%, 25%, 50% titrations: 11%, 21%, 44%; Spearman=0.94; p<0.001) and NS (11%, 28%, 58%; Spearman=0.790; p<0.001). However, HBOC also enhanced fibrinolysis without the addition of tPA (1%,4%,5%; Spearman=0.735; p=0.001) and NS did not (1%,2%,1%; r=0.300; p=0.186; Figure 1).  Moreover, addition of TXA did not alter or inhibit this fibrinolysis (WB vs 50% HBOC: 1.8% vs 5.65%, p=0.04).  There was no significant difference in fibrinolysis of HBOC with or without TXA (50% HBOC vs 50% HBOC +TXA: 5.55% vs 5.65%, p=0.92).  Additionally, the increased fibrinolysis seen with NS was reversed when TXA was present (WB vs 50% NS: 1.8% vs 1.65%, p=1.0).

Conclusion: HBOCs enhance fibrinolysis without the addition of tPA; moreover, this is independent of plasmin as the phenomenon persists in the presence of TXA.  Our findings implicate the hemoglobin molecule or its components in stimulating fibrinolysis

 

3.04 Traumatic Brain Injury Alters Circulating Microparticles which then Impact Coagulation

Posted on December 22, 2014

E. F. Midura1, C. C. Caldwell1, M. D. Goodman1  1University Of Cincinnati,Cincinnati, OH, USA

Introduction:  Traumatic brain injury (TBI) is known to result in a sub-acute, post-traumatic, hypercoagulable state, however the pathophysiology behind this alteration is poorly understood. Platelet-based contribution to clot is known to decrease over time following trauma and microparticles have also been shown to be changed after TBI. Whether platelets and microparticles interact to influence coagulation has not been determined. Using a murine model, we hypothesized that microparticle and platelet contributions to clot formation would be altered after TBI. 

Methods:  An established weight-drop model was used to induce TBI in anesthetized mice. Sham mice underwent anesthesia without TBI. Blood samples were collected 24 hours after injury, and circulating microparticle and platelet counts determined. Thromboelastometry (ROTEM) was used to evaluate changes in hemostasis. ROTEM was then repeated on sham and post-injury blood treated with normalized concentrations of microparticles isolated from sham mice (sham microparticles), TBI mice (TBI microparticles), or microparticle-free saline. A microparticle pro-coagulant assay was used to compare activity in sham and TBI mice.

Results: One day after TBI, we observed a decrease in overall microparticle concentrations (3.6 vs. 1.9 x 108 microparticles/ml, p < 0.001). Overall platelet counts did not change after injury. ROTEM demonstrated a reduced platelet contribution to hemostasis 24 hours after TBI compared to sham (62.9% vs. 48.0%, p < 0.01). When TBI microparticles were added to sham blood, a significant decrease in platelet contribution to clot was seen compared to saline (Figure 1). Conversely, when sham microparticles were added to blood from injured mice, there was a normalization of platelet contribution to clot formation when compared to saline (Figure 1). Furthermore, when microparticle pro-coagulant activity was assayed, TBI microparticles had increased activity compared to sham microparticles (3.28 vs. 1.72, p = 0.04).

Conclusion: Our data demonstrate that after TBI, 1) circulating microparticles are decreased, 2) platelet contribution to clot formation is decreased despite unchanged platelet count, and 3) TBI microparticles independently augment post-traumatic clot formation. Post-TBI microparticles may therefore be responsible for the altered platelet role in coagulation and the development of a post-traumatic hypercoagulable state.

 

26.10 Heterotopic Ossification is Inhibited in Leptin-deficient (ob/ob) Mice Despite Robust Vessel Growth

Posted on December 22, 2014

S. Agarwal1, J. Peterson1, O. Eboda1, S. Loder1, C. Brownley1, A. Donneys1, D. Fine1, K. Stettnichs1, K. Ranganathan1, S. Wang1, S. Buchman1, P. Cederna1, B. Levi1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction:  Clinical and laboratory experience has shown that diabetes contributes to delayed wound and bony healing. However, the relationship between diabetes and the generation of heterotopic ossification (HO) following trauma has not been previously studied. By understanding how diabetes potentiates ectopic bone formation, we may be able to identify treatment strategies which target similar local or systemic factors to prevent HO in patients following trauma, large surface-area burns, and surgical procedures.

Methods:  Male leptin-deficient (ob/ob) or wild type mice (C57BL/6 background) underwent 30% total body surface area burn injury with left hind limb Achille’s tenotomy.  At 7 weeks, mice were injected with Microfil contrast and hind limbs were imaged with micro-CT to quantify aggregate vessel volume.  Vessel volumes were normalized to hind limb mass to account for differences in limb size between diabetic and wild type mice.  A second set of mice underwent micro-CT every 2 weeks up to 9 weeks to quantify HO volume (Hounsfield unit threshold 1000) after adjusting images to remove normal tibia and fibula bone.  

Results: HO volume in diabetic mice was significantly lower than in wild type mice at 9 weeks following burn and tenotomy injury (3.99 mm3 v. 6.20 mm3, p<0.01). Diabetic mice exhibited a decreasing trend in HO volume from week 5 to week 9 based on micro-CT (r=-0.52, p =0.15), suggesting that HO had actually resorbed in these mice.  The mean vessel volume in the hind limbs of diabetic mice 7 weeks after burn and tenotomy was significantly greater than the mean vessel volume in wild type mice (20.6 mm3 v. 4.7 mm3, p <0.05).  When adjusting for limb mass, we found that diabetic legs continued to have more vessel volume (14.1 mm^3/g v. 7.2 mm3/g).  Further examination of vessel architecture showed that the diabetic hind limbs had small vessel growth with disorganized character, while the wild type hind limbs had fewer, but larger vessels.

Conclusion: Here we use Microfil with micro-CT imaging to compare vessel formation between diabetic (leptin-deficient) and wild-type mice in a model of ectopic bone formation. Our findings demonstrate that despite robust small vessel formation at the site of hind limb trauma, diabetic mice produce less ectopic bone than wild type mice.  We also found that diabetic mice actually exhibit resorption of ectopic, suggesting that these mice may be unable to sustain bone growth. This may be related to the disorganized pattern of vessel growth characteristic of the wounds in diabetic mice, as demonstrate by Microfil.  Our findings may elucidate additional targets, such as the local vascular milieu, to prevent initial ectopic bone growth or recurrence following surgical excision.

27.10 ORGAN-SPECIFIC DIFFERENCES IN FIBRINOLYSIS DURING ISCHEMIA

Posted on December 22, 2014

E. Gonzalez1, H. B. Moore1, M. P. Chapman1, M. Fragoso1, A. P. Morton1, C. C. Silliman1,2, A. Banerjee1, E. E. Moore1,3  1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2Bonfils Blood Center,Department Of Researcj,Denver, CO, USA 3Denver Health Medical Center,Department Of Surgery,Denver, CO, USA

Introduction:  The fibrinolytic response to trauma and hemorrhagic shock has been thus far characterized as a hyper-fibrinolytic state, which exacerbates coagulopathic bleeding. However, recently we have identified a subpopulation of patients with impaired fibrinolysis, also referred to as fibrinolysis shutdown, which favors unregulated clotting, particularly in the microvasculature. This spectrum of fibrinolysis may occur with simultaneous heterogeneity throughout tissue beds. We hypothesized that the response to ischemia is organ specific, with distinct patterns of fibrinolysis. 

Methods:  Six Yorkshire swine were anesthetized, and after laparotomy, the kidney, small bowel, liver, and spleen were subjected to controlled ischemia by arterial clamping. Venous samples were obtained from these organs at baseline (before ischemia), then at 15 and 30 min. after clamping, and 5 min. after reperfusion. A systemic sample from the saphenous vein was also obtained at theses time points. A systemic mean arterial pressure of 35 mmHg was induced after clamping during this period of time. Thrombelastography (TEG) was performed from re-calcified citrated samples between 20-40 min. from collection. Changes in TEG variables for each organ along these time-points were compared by the Friedman test for significance (p<0.05), with post-hoc pairwise comparisons (Bonferroni adjusted p-value).
 

Results: The TEG variables R-time, alpha-angle, and maximal amplitude (MA), all progressively demonstrated a coagulopathic profile with ischemia time, and did not normalize after reperfusion, for all organs and for the systemic venous samples. Fibrinolysis quantified by TEG (%LY30: percent of clot lysis 30 min. after reaching MA) progressively decreased with ischemia, demonstrating fibrinolysis shutdown; this was statistically significant for the kidney and liver (Fig. 1). However, the systemic venous samples demonstrated hyper-fibrinolysis proportional to ischemia time, which was also statistically significant. All organs had statistically significantly less fibrinolysis than the systemic venous sample at 15 min. and 30 min. of ischemia, and after reperfusion (Fig. 1).

Conclusion:  Despite evidence of systemic hyper-fibrinolysis during shock, paradoxically, fibrinolysis shutdown occurs simultaneously at an organ level, particularly in the kidney and liver. Consequently, treatment of hyperfibrinolysis with anti-fibrinolytics, such as tranexamic acid, may have adverse effects in certain organs’ microvasculature. These differences in the spectrum of fibrinolysis provide further insight into the pathophysiology of trauma-induced coagulopathy, allowing for study of its optimal diagnosis and management. 

 

24.08 Selective inhibition of Histone Deacetylase 6 Improves Survival in a Lethal Two-hit Model

Posted on December 22, 2014

X. Cheng1, Z. Liu1, B. Liu1, Y. Li1, H. B. Alam1  1University Of Michigan,General Surgery,Ann Arbor, MI, USA

Introduction: Hemmorrhagic shock (HS) followed by a subsequent infection (“second hit”) is highly lethal. We have previously demonstrated that treatment with valproic acid, a pan histone deacetylase (HDAC) inhibitor, improves survival in a rodent “two-hit” model. In the present study, our aim was to determine whether selective inhibition of histone deacetylase 6 with Tubstatin A (Tub A) could prolong survival in a 2-hit model.

Methods: C57Bl/6J mice were subjected to HS (40% blood loss) and then randomly divided into 2 groups (n=13/group): The treatment group was injected (intraperitoneal) with Tub A (70 mg/kg) dissolved in 1 µl/g of dimethyl sulfoxide (DMSO), whereas the vehicle (Veh) group was given 1 µl/g of DMSO. After 24 h, all mice were subjected to cecal ligation and puncture (CLP) followed by a second dose of Tub A or DMSO. Survival was monitored for 10 days. In a parallel study, peritoneal irrigation fluid and liver tissues were collected at 3h after CLP.  Enzyme-linked immunosorbent assay was performed to determine the myeloperoxidase (MPO) activity (marker of  neutrophil activation) and TNF-α and IL-6 concentrations in the peritoneal irrigation fluid, whereas real-time PCR was performed to measure the relative mRNA levels of TNF-α and IL-6 in the liver tissue.

Results: Tub A administration significantly improved survival compared to the control (69.2% vs 15.4%, p<0.0074) (Figure). In addition, Tub A significantly suppressed MPO activity (169.9±8.4 ng/ml vs 70.4±17.4ng/ml; p=0.0001), and inhibited levels of cytokine TNF-α and IL-6 in the peritoneal fluid (TNF-α: 105.7± 4.7 pg/ml vs 7.4±2.4 pg/ml; IL-6: 907.4±2.3 pg/ml vs 483.6 ±1.6 pg/ml; p=0.0001) compared to the vehicle control. Meanwhile, the hepatic gene expression of these cytokines was significantly lower (TNF-α: 18.9±7.8 vs 0.92±0.25; IL-6: 43.3±5.8 vs 8.3±3.4; p=0.0001) in the Tub A treated animals.

Conclusion: Tub A treatment significantly improves survival, attenuates inflammation and down regulates the gene expression and protein levels of TNF-α and IL-6 in a rodent two-hit model.

 

24.09 Selective Inhibition of SIRT2 improves Outcomes in a Lethal Septic Model

Posted on December 22, 2014

T. Zhao1, Y. Li1, B. Liu1, R. T. Bronson2, H. B. Alam1  2Harvard School Of Medicine,Brookline, MA, USA 1University Of Michigan,General Surgery,Ann Arbor, MI, USA

Introduction: Six isoforms of histone deacetylase (HDAC) Class III have been reported- Sirtuin (SIRT) 1, 2, 3, 4, 5, and 6. We recently demonstrated that EX-527, an inhibitor of SIRT1, improves survival in a lethal cecal ligation and puncture (CLP) model. The aim of this study was to determine if selective inhibition of SIRT2 with AGK2 could also improve survival in a lethal septic model, and attenuate the inflammatory response.

Methods: Experiment I: C57BL/6J mice were intraperitoneally injected with either AGK2 (82 mg/kg) dissolved in dimethyl sulfoxide (DMSO) or DMSO only, and 2 h later subjected to CLP (n=9/group). Survival was monitored for 10 days. Experiment II: animals, treated the same way as Experiment I, were grouped into (i) DMSO vehicle, and (ii) AGK2 (n=10-14/group), with sham-operated animals serving as controls. Peritoneal fluid and blood samples were collected for measurement of cytokines at 24 or 48 h. Blood at 48 h was also used to assess the coagulation status using Thrombelastography (TEG). In addition, long bones (femurs and tibias) were harvested (n=5-6/group) at 48 h to determine morphological changes in the bone marrow by H&E staining. Bone marrow atrophy was quantified by a blinded pathologist. Experiment III: Normal primary splenocytes were cultured, and treated with lipopolysaccharide (LPS) in the presence or absence of AGK2 (10 μM) for 6 h to assess cytokine production (n=4/group).

Results: AGK2 significantly improved survival (figure), and attenuated the levels of cytokines in the circulation (TNF-α: 298.3±24.6 vs. 26.8±2.8 pg/ml, p=0.003; IL-6: 633.4±82.8 vs. 232.6±133 pg/ml, p=0.034) and peritoneal fluid (IL-6: 704.8±67.7 vs. 391.4±98.5 pg/ml, p=0.033) compared to the vehicle control. It also decreased the TNF-α and IL-6 production by the splenocytes in-vitro (TNF-α: 68.1±6.4 vs. 23.9±2.8 pg/ml, p=0.001; IL-6: 73.1±4.2 vs. 49.6±3.0 pg/ml; p=0.005). The TEG data showed that animals subjected to CLP displayed markers of coagulopathy: prolonged fibrin formation and fibrin cross-linkage time, slower clot formation, decreased platelet function and clot rigidity. Inhibition of SIRT2 was associated with dramatic improvements in fibrin cross-linkage, platelet function and clot rigidity, but without a significant impact on the clot initiation parameters. Meanwhile, inhibition of SIRT2 significantly attenuated the bone marrow atrophy (58.3±6.5 vs. 30.0±8.2%, p=0.026).

Conclusions: Selective inhibition of SIRT2 significantly improves survival, attenuates “cytokine storm” and sepsis-associated coagulopathy, and decreases bone marrow atrophy in a lethal septic model.

 

25.01 Carbon Monoxide Protects Against Mitochondrial Injury Following Hemorrhage and Resuscitation

Posted on December 22, 2014

P. Waltz1, J. Luclano1, S. Shiva1, B. Zuckerbraun1,2  1University Of Pittsburg,Pittsburgh, PA, USA 2VA Pittsburgh Healthcare System,Pittsburgh, PA, USA

Introduction:  Currently, there is no effective resuscitative adjunct to fluid and blood products to limit tissue injury for traumatic hemorrhagic shock.  The objective of this study was to investigate the role of inhaled carbon monoxide (CO) to limit inflammation and tissue injury, and specifically mitochondrial damage, in experimental models of hemorrhage and resuscitation.

Methods:  Mice underwent hemorrhagic shock and resuscitation to a mean arterial pressure (MAP) of 20 or 25 mmHg.  Mice were resuscitated with Lactated Ringers (2:1 volume of maximal shed blood ) after  90 or 120 minutes of hypotension.  Pigs (30-35 kg) were anesthetized and bled to a MAP of 30-40 mmHg for 90 minutes, followed by initial resuscitation with Hextend (1:1 volume of shed blood).  Pigs had ongoing resuscitation and support for up to four hours.  Data were collected continuously.  Primary mouse hepatocytes were used for in vitro studies and murine skeletal muscle was used for ex vivo studies.  ANOVA was used for statistical analysis and significance was assumed with a P<0.05.

Results:Inhaled CO (250 ppm for 30 minutes) protected against mortality in severe murine hemorrhagic shock and resuscitation (HS/R) (20% vs. 80%; P<0.01).  Additionally, CO limited the development of shock as determined by arterial blood pH (7.25±0.06 vs. 7.05±0.05; P<0.05), lactate levels (7.2±5.1 vs 13.3±6.0; P<0.05), and base deficit (13±3.0 vs 24±3.1; P<0.05).  A dose response of CO (25-500 ppm) demonstrated protection against HS/R lung and liver injury as determined by MPO activity and serum ALT, respectively.  CO limited HS/R-induced increases in serum tumor necrosis factor-α and interleukin-6 levels as determined by ELISA (P<0.05 for doses of 100-500ppm).  Furthermore, inhaled CO limited HS/R induced oxidative stress as determined by hepatic oxidized glutathione:reduced glutathione levels and lipid peroxidation.  In porcine HS/R, CO did not influence hemodynamics. However,  CO limited HS/R-induced skeletal muscle and platelet mitochondrial injury as determined by respiratory control ratio (muscle) and ATP-linked respiration and mitochondrial reserve capacity (platelets).  Furthermore, in vitro and ex vivo, CO limited oxygen consumption to increase oxygen levels under hypoxic conditions, and also limited loss of mtiochondrial membrane potential, oxidative stress, and loss of ATP.

Conclusion:  CO therapy limits injury to the metabolic machinery of cells in the setting of HS/R in multiple animal models. These preclinical studies suggest that inhaled CO can be a protective therapy in HS/R, however, further clinical studies are warranted. 

25.02 Genetic Modulation to Improve Traumatic Brain Injury Induced Inflammation

Posted on December 22, 2014

J. Lee1, K. Sail1, T. Costantini1, B. Eliceiri1, R. Coimbra1, V. Bansal1  1University Of California – San Diego,Department Of Surgery, Division Of Trauma, Surgical Critical Care And Burns,San Diego, CA, USA

Introduction: The invasion of leukocytes following traumatic brain injury (TBI) may have significant consequences. Excessive leukocytes are not beneficial and instead may cause an outsized inflammatory response initiating secondary neuronal injury and exacerbating long-term cognitive dysfunction.  Therefore, modulating leukocyte migration may lead to novel therapy.  Previously, we have shown that Esophageal Cancer Related Gene 4 (Ecrg4) is a novel leukocyte chemoattractant in glioblastoma. We hypothesize that Ecrg4 is a potent mediator of TBI induced leukocyte recruitment.

Methods: A controlled cortical impact model was used to create moderate TBI in 12 wk old wild type (WT) or Ecrg4 knock-out (KO) mice. Three days post-TBI, cells were isolated and flow cytometry analysis was used gating for microglia (CD45lowCD11b+ cells), monocytes (CD45highCD11b+ cells), and lymphocytes (CD45highCD11b- cells). Brain sections were immunostained with Iba-1 and CD11b to assess recruitment and activation of microglia and migrating leukocytes. Brain homogenates were collected following TBI and cytokine levels (IL-12p70, TNF, IFN-γ , MCP-1, IL-10, and IL-6) were measured by multiplex enzyme-linked immunosorbent assay (ELISA).

Results: TBI resulted in a rapid invasion of activated microglia and inflammatory monocytes, peaking at post-TBI Day 3 and persisting at Day 7 (19 ±30 vs. 1018 ± 694; p<0.05). Invading cells expressed surface markers for activation (MHCII+ and CD86+). Ecrg4 KO mice demonstrated reduced infiltration of microglia (101 ±694 vs. 299 ±143; p<0.05) and inflammatory monocytes (314 ±155 vs. 163 ±118; p<0.05) compared with Ecrg4 WT after TBI. Activated microglia (471 ±326 vs. 125 ±63; p<0.05) were also reduced in Ecrg4 KO mice when compared to Ecrg4 WT mice.  TBI induced an increase in pro-inflammatory mediators (TNF, MCP-1, and IL-6), peaking at six hours post-TBI.  TNF levels in the brain were significantly decreased in Ecrg4 KO mice compared with Ecrg4 WT mice (31± 12 vs. 4±2pg/ml; p<0.05) following TBI.

Conclusion: Genetically deficient Ecrg4 mice had altered leukocyte invasion and microglial activation following TBI, confirming Ecrg4’s role as a novel chemoattractant. Future studies will assess whether Ecrg4-mediated leukocyte invasion alters post-TBI histopathology and neurocognitive recovery.

 

25.03 DPR Reduces Visceral Ischemia and Inflammatory Cytokines Following Hemorrhagic Shock

Posted on December 22, 2014

J. L. Weaver1, S. Matheson1, P. Matheson1, C. Downard1, R. N. Garrison1, J. Smith1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction: Hemorrhagic shock (HS) is a significant cause of morbidity and mortality in trauma patients. Treatment has traditionally been intravenous fluids and blood products to restore intravascular volume. However, studies have shown that even after blood pressure normalizes, the visceral organs remain ischemic. This can lead to organ dysfunction, failure, and death.  This prolonged ischemia is thought to be due in part to the activation of an inflammatory cascade of cytokines. Direct peritoneal resuscitation (DPR) improves blood flow to the abdominal organs. In this study we examine the effect of DPR on the body’s inflammatory response to hypotensive shock.

Methods: Rats were randomly assigned to three groups (n=8/group): 1) sham (no HS); 2) HS plus conventional resuscitation (CR); or 3) HS+CR+DPR.  Rats were hemorrhaged to 40% MAP and resuscitated with shed blood and two volumes of normal saline. Group 3 rats were given intraperitoneal injections of 30cc 2.5% peritoneal dialysis solution. Effective hepatic blood flow (EHBF) was measured using a steady state of galactose. Tissue was collected at 4 hours post-resuscitation.  Serum cytokines were measured using the Luminex immunoassay or ELISA.

Results: Central hemodynamics were restored post-hemorrhage in both groups, but EHBF fell after resuscitation was complete. This was prevented by adding DPR. The DPR groups had lower serum levels of hyaluronic acid (HA) and high-mobility group box-1 protein (HMGB-1), which are markers of cellular injury, when compared to the CR group. HMGB-1 is usually sequestered in the cell’s nucleus and immunohistochemistry staining shows this is maintained in the DPR group, but the nucleus is disrupted and HMGB-1 migrated to the cytosol in the CR group. The DPR group showed lower levels of inflammatory cytokines such as IL-1a, IL-1B, IL-6 and INF-γ. Histologic sections of terminal ileum in the DPR rats also demonstrated better preservation of architecture with less edema and necrosis.

Conclusion: The addition of DPR after HS improved visceral blood flow, demonstrated by the EHBF data. This led to a preservation of cell structure, seen on histology and via a reduction in HA and HMGB-1, which are released in cell necrosis. HMGB-1 is normally stored in the nucleus during apoptosis to prevent inflammatory signaling. It was released to the cytosol in the CR group but not the DPR group, which further proves worse necrosis with CR. DPR also returned levels of inflammatory cytokines to near-normal when compared to the CR group. These findings suggest that DPR reduces the inflammatory response which contributes to multiple organ-system failure after hemorrhagic shock, and has the potential to improve outcomes in trauma patients.

25.04 Age-Dependent Changes of Metabolic Pathways in the Lung Following Murine Hemorrhagic Shock

Posted on December 22, 2014

L. R. Klingbeil1,2, G. Piraino1, P. W. Hake1, J. R. Ledford1, B. Zingarelli1  1Cincinnati Children’s Hospital Medical Center,Department Of Critical Care,Cincinnati, OH, USA 2University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA

Introduction:  Despite advances in the management of trauma victims, mortality from hemorrhagic shock and the ensuing multiple organ dysfunction syndrome (MODS), remains high. Pediatric patients have a lower incidence of MODS, including acute lung injury, than adult patients. The molecular mechanisms underlying this age-dependent susceptibility to MODS are not fully understood. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which controls autophagy and metabolic recovery through mitochondrial biogenesis. Following activation by phosphorylation, AMPK regulates mitochondrial biogenesis via peroxisome proliferator-activated receptor γ co-activator α (PGC1-α), which can also be regulated by sirtuin 1(Sirt-1). We hypothesize that these metabolic signaling pathways are altered in the lung during hemorrhagic shock and are age-dependent.

Methods:  Hemorrhagic shock was induced in anesthetized young male (2-4 months old) and mature mice (9-10 months old) by withdrawing blood from the femoral artery to a mean arterial pressure (MAP) of 30 mmHg for 90 minutes. The mice were then resuscitated with the shed blood plus two times that amount in Lactated Ringer’s solution. Mice were sacrificed 3 hours after resuscitation and lungs were harvested for biochemical assays. Sham mice underwent a similar surgical preparation but were not bled.

Results: After hemorrhagic shock, there was marked neutrophil infiltration, as evaluated by myeloperoxidase (MPO) assay, in the lung of young mice when compared to the sham mice (201.3±19.4 versus 132.9±17.7 U/100mg tissue, p<0.05) indicating increased inflammation after hemorrhage. Mature mice also exhibited higher neutrophil infiltration in the lung after hemorrhagic shock when compared to mature sham mice (222.1±30.1 versus 167.6±12.9 U/100mg tissue, p<0.05). At western blot analysis, there was a significant increase of nuclear pAMPK (1.7±0.06 relative intensity) in lung of young mice after hemorrhagic shock when compared to age-matched sham mice (1.0±0.04 relative intensity, p<0.05) and was associated with maintenance of normal levels of PGC1-α and Sirt-1, thus suggesting the capability to mount a metabolic response. Interestingly, nuclear levels of pAMPK were significantly increased in sham mature mice (2.2±0.01 relative intensity) when compared with young sham animals (p<0.05). However, after hemorrhagic shock in lung of mature mice, there was a significant decrease in pAMPK expression (1.2±0.21 relative intensity), which was associated with a marked down-regulation of PGC1-α and Sirt-1 when compared to sham age-matched mice, thus suggesting the impairment to promote a metabolic recovery after stress.

Conclusion: Our data suggests that during hemorrhagic shock, metabolic repair mechanisms are activated in the lung and involve AMPK-dependent pathways. However, this restorative process diminishes in old age.

25.05 IL-17A/F is Associated with the Immunopathology of Trauma in Humans and Mice

Posted on December 22, 2014

M. H. Ramadan1, R. Namas1, Y. Vodovotz1, T. R. Billiar1  1University Of Pittsburg,Surgery,Pittsburgh, PA, USA

Introduction:

Excessive and sustained inflammation is associated with persistent critical illness and complications such as organ dysfunction and nosocomial infection in blunt trauma patients.  The roles of many proximal mediators of trauma induced inflammation, such as IL-6 and MCP-1 are well studied.  However, the involvement of key downstream mediators including IL-17/F remains unknown.  IL-17A/F, which can be produced by Th17 cells or certain populations of innate lymphoid cells, can promote sustained inflammation and organ injury in both acute and chronic conditions.  Here, we hypothesized that IL-17A/F levels would correlate with a complicated course in human trauma patients and organ injury in mice subjected to hemorrhagic shock and trauma (HS/T).

Methods:

Animal experiments: Anesthetized C57BL/6 mice were subjected to bilateral lower extremity injury and pressure-targeted sever hemorrhagic shock (HS) with mean BP 25-30mmHg, monitored for 2 hours via bilateral femoral cannulation. Animals were then resuscitated with X3 volume of shed blood with lactated Ringer’s solution. One group of mice received IL-17-blocking antibody 12 hours prior to HS and re-dosed at the time of resuscitation. Equal volume of PBS was administered to the mice in the control group. Plasma IL-6, MCP-1 levels and ALT were measured at 6 hours (n=6/group).

Human Samples: Plasma IL-17A/F levels were measured in the serum of 88 consented, severely injured (average ISS=26, 44 patients with a complicated course and 44 with an uncomplicated course) blunt trauma patients upon admission and over time. The two cohorts were matched for ISS, age and sex.

Results:

The human data showed elevated IL-17A/F Levels in the complicated patient group; those levels were elevated within the first 24 hours of injury. These levels remained elevated and significantly higher than the uncomplicated patient group even prior to the development of infectious complications.

In the mice we were able to demonstrate significantly elevated plasma IL-17A/F levels that peaked at 6 hours after trauma correlating with peak organ damage. There were no baseline differences between the antibody treated mice and the controls. Both the treatment and control groups had similar plasma IL-6 and MCP-1 levels indicating and equivalent proximal inflammatory responses. The Blocking of IL-17 lead to a significant reduction in liver damage, measured by 62% lower ALT p= 0.007 in the antibody treatment group; supporting our hypothesis.

Conclusion:

Our clinical data show that IL-17A/F levels correlate with persistent critical illness in human trauma patients while our mechanistic studies in a mouse polytrauma model confirms that IL-17A participates in organ damage as a mediator downstream of IL-6.  These data also suggest that IL-17 producing lymphocyte populations become activated early in clinical and experimental trauma.

25.06 Fibrinolysis Shutdown Phenotype Masks Changes in Rodent Coagulation Measured by Thrombelastography

Posted on December 22, 2014

H. B. Moore1, P. Lawson1, M. Fragoso1, E. Gonzalez1, M. P. Chapman1, F. Gamboni1, S. Mitra1, C. A. Anderson1, A. Banerjee1, C. C. Silliman1, E. E. Moore1  1University Of Colorado,Surgery,Denver, COLORADO, USA

Introduction: The use of thrombelastography(TEG) in trauma has identified unique phenotypes of coagulopathy. Pathologic hyperfibrinolysis (excessive blood clot degradation) and fibrinolysis shutdown (impaired blood clot degradation) are associated with increased mortality compared to a low level of physiologic fibrinolysis. Investigating the regulation of fibrinolysis with animal models has been challenging and has low yield to clinical translation. We hypothesize that rats have a baseline fibrinolysis shutdown phenotype and require an exogenous challenge of a profibrinolytic to differentiate mechanisms that promote or inhibit fibrinolysis. 

 

Methods: Fibrinolysis resistance was assessed by TEG with exogenous tissue plasminogen activator(tPA) titrations in rat whole blood. The experimental groups(n=9 per group) were: 1)tissue injury (laparotomy/bowel crush), 2) shock(hemorrhage to MAP < 25 mmHG), and 3) sham(arterial canulation and tracheostomy).  Blood was sampled at 30 min, and assayed with native TEG challenged with taurcholic acid(TUCA, a known inhibitor of anti-fibrinolytic protein).  The percent of remaining clot strength 30 min after reaching maximum amplitude(CL30) was used to measure clot resistance to fibrinolysis.  Plasma was assayed for tPA concentration.  Liver histology was assessed for organ damage due to oxygen sensitivity and receiving direct portal blood supply.

 

Results: Rat blood was resistant to exogenous tPA, CL30 at 150ng/ml(p=0.511) and 300ng/ml(p=0.931) was similar to baseline, while 600ng/ml(p=0.046) provoked fibrinolysis.  Baseline(p=0.679) and post-procedure CL30(p=0.505) were not significantly different between groups when using a native TEG.  TEG TUCA challenge showed no differences at baseline CL30(p=0.937); whereas, post-procedure there were differences in CL30 (Fig 1, p<0.001). This corresponded to similar plasma tPA concentrations(p=0.423) at baseline between rats and different tPA concentration(p=0.003) post procedure. The percent change in CL30 from baseline was increased in tissue injury compared to sham(p=0.048.); whereas, CL30 decreased in shock versus sham(p=0.048).  Total tPA concentration was higher in the shock group compared to trauma(p=0.009) and sham(p=0.012). Histologic evidence of cellular damage was pronounced in trauma and shock compared to baseline. 


 

Conclusion:  The TEG TUCA challenge is capable of differentiating changes in clot stability with rats undergoing diverse stresses. Tissue injury inhibits fibrinolysis while shock promotes tPA mediated fibrinolysis, consistent with our recent clinical studies.  This technical modification of TEG may be valuable for elucidating the mechanisms responsible for the spectrum of fibrinolysis observed following severe trauma.

25.07 Regulation of Hypoxic Alveolar Epithelial Cells in Lung Contusion

Posted on December 22, 2014

M. Sherman1, M. V. Suresh1, D. Machado-Aranda1, B. Thomas1, N. Talarico1, Y. Shah2, K. Raghavendran1  1University Of Michigan,Acute Care Surgery/General Surgery,Ann Arbor, MI, USA 2University Of Michigan,Molecular Biology And Integrative Physiology,Ann Arbor, MI, USA

Introduction: Lung contusion (LC) due to major thoracic trauma is a significant risk factor for the development of Acute Respiratory Distress Syndrome. The main pathological consequence of LC is hypoxia. The key mediator of adaptation to hypoxia is hypoxia-inducible factor (HIF)-1. We have recently published that a key driver of acute inflammation following LC is HIF-1α. This is based on significant reductions in lung injury and inflammation observed in type II alveolar epithelial cells (AEC) of HIF-1α conditional knockout mice. To better understand the regulation of hypoxic AEC, the current project evaluated the fate of hypoxic AEC following LC.

Methods: LC was induced using a cortical contusion impactor in WT C57Bl6 and ODD-Luc (oxygen dependent domain of HIF linked to luciferase) mice. In the WT mice, global hypoxia was confirmed by pimonidazole staining after LC. In the ODD-Luc mice, subsequent IVIS imaging was performed to assess hypoxia. To evaluate the fate of hypoxic AEC (high luciferase activity), dual staining with caspase-3, TUNEL staining (apoptosis), and Ki67 (proliferation marker) were performed. Finally, laser capture microdissection (LCM) was used to isolate hypoxic and normoxic cells after LC at 24 h, yielding adequate quantities of RNA that were subjected to real-time PCR.

Results: Using Hypoxyprobe (pimonidazole), the presence of global hypoxia with LC was confirmed in the WT mice. Lung, liver, and spleen samples ODD-Luc mice showed increased luminescence at all-time points after LC. Presence of hypoxia in AEC was not uniform.  Most of the AEC that were hypoxic following LC showed evidence of caspase-3 activation. Similar results were observed with TUNEL staining. Also, the hypoxic AEC did not show a significant increase in proliferation (ki67). Using directly conjugated fluorescent-tagged luciferase antibody, hypoxic cells were visualized and isolated by LCM. Figure 1 shows the representative immuno-fluorescent staining for luciferase positive hypoxic cells (red circles, Luc+) and the adjacent normoxic cells (blue circles, Luc-) in lung samples in ODD-Luc mice 24 h after LC as well as before and after LCM. Isolated RNA also showed significant increases in VEGFa and caspase-1 in hypoxic AEC compared to normoxic AEC.

Conclusion: Hypoxic AEC following LC specifically undergo apoptosis. Moreover, the hypoxic AEC have increased VEGFa and caspase-1 activation, which confirms our previous finding that HIF-1α regulation of AEC is likely mediated through inflammasome activation.

 

25.08 Hydrogen Inhalation Protects Against Acute Lung Injury Induced by Hemorrhagic Shock/ Resuscitation

Posted on December 22, 2014

K. KOHAMA1, H. Yamashita1, M. A. Ishikawa1, T. Nishimura1, N. Fujisaki1, J. Kotani1, A. Nakao1  1Hyogo College Of Medicine,Department Of Emergency, Disaster And Critical Care Medicine,Nishinomiya, HYOGO, Japan

Introduction: Hemorrhagic shock followed by fluid resuscitation (HS/R) triggers an inflammatory response characterized by upregulation of proinflammatory cytokines and adhesion molecules and induces pulmonary inflammation that leads to acute lung injury (ALI). Hydrogen, a novel therapeutic gas, has potent cytoprotective, anti-inflammatory, and antioxidant effects. This study aimed to examine the effects of inhaled hydrogen gas on lung injury caused by HS/R.

Methods: Sprague-Dawley rats were subjected to hemorrhagic shock by withdrawing blood to achieve a mean arterial pressure of 30±5 mm Hg for 60 minutes followed by resuscitation with shed blood and saline, as needed, to restore blood pressure. After HS/R, the rats were either maintained in room air or were exposed to 1.3% hydrogen (1.3% H2, 21% O2, 77.7% N2) in a gas-exposure chamber. Rats were randomly assigned to three experimental groups consisted of sham, HS/R (air) and HS/R (hydrogen).

Results: HS/R induced ALI, as demonstrated by significantly impaired gas exchange as well as congestion, edema, cellular infiltration, and hemorrhage in the lungs. Hydrogen inhalation mitigated lung injury, as indicated by significantly improved gas exchange 3 hours after resuscitation, and reduced congestion, edema, cellular infiltration, and hemorrhage. Hydrogen inhalation did not affect hemodynamic status or tissue oxygenation during HS/R. HS/R causes the rapid production and release of several proinflammatory mediators and induces epithelial apoptosis associated with neutrophil sequestration in the lung. Exposure to 1.3% hydrogen significantly attenuated the upregulation of the mRNAs for several proinflammatory mediators (tumor necrosis factor (TNF)-α, interleukin-6 and intercellular adhesion molecule (ICAM)-1) and reduced epithelial apoptosis. Lipid peroxidation, determined by measurement of tissue malondialdehyde (MDA) levels, was significantly reduced in the presence of hydrogen, indicating antioxidant effects.

Conclusions: Hydrogen, administered through inhalation, may exert potent therapeutic effects against ALI induced by HS/R and attenuate activation of inflammatory cascades.

25.09 The alpha-7 nicotinic acetylcholine receptor mediates the vagal anti-inflammatory response to injury

Posted on December 22, 2014

S. Langness1, B. P. Eliceiri1, V. Bansal1, R. Coimbra1, T. W. Costantini1  1University Of California – San Diego,Division Of Trauma, Surgical Critical Care, Burns, And Acute Care Surgery,San Diego, CA, USA

Introduction:  Acute lung injury is a frequent complication of severe burns and is responsible for approximately 10-15% of burn-related deaths. Acute lung injury occurs as a result of an unrestrained immune response after injury and is understood to be initiated by gut barrier failure and gut inflammation. We have previously shown that vagus nerve signaling (VNS) limits burn-induced acute lung injury through its ability to prevent gut barrier failure. While the alpha-7 nicotinic acetylcholine receptor (alpha-7 nAChR) is required to mediate the systemic anti-inflammatory effects of VNS in the spleen, the role of the alpha-7 nAChR in limiting acute lung injury via the gut-lung axis is unknown. We hypothesized that the alpha-7 nAChR is required for vagal-mediated lung protection, where the anti-inflammatory effects of VNS would be lost in alpha-7 nAChR KO mice.

Methods:  Male alpha-7 nAChR knock-out (KO) and matched control C57BL/6 wild-type (WT) mice were subjected to 30% total body surface area cutaneous burn. A separate cohort of WT and KO animals was treated with cervical VNS following injury.  Changes in gut architecture were characterized using histology.  Lung injury was evaluated by histology and by measuring changes in inflammatory cell infiltration using florescence-labeled antibodies to myeloperoxidase (MPO) and CD68, surrogate markers for neutrophils and macrophages, respectively.

Results:  Severe burn caused histologic gut and lung injury in both alpha-7 nAchR WT and KO mice.  Burn-induced lung injury was characterized by increased staining for MPO and CD68 compared to sham.  VNS prevented burn-induced gut barrier injury and lung inflammation in alpha-7 nAchR WT mice. The protective effects of VNS on gut barrier failure after injury were lost in alpha-7 nAchR KO mice. VNS also failed to prevent acute lung injury in burn-injured alpha-7 nAchR KO mice, with lung histology similar to alpha-7 nAchR WT and KO mice exposed to burn alone.  The ability of VNS to attenuate the mobilization of inflammatory cells to the lung after burn injury was also lost in alpha-7 nAchR KO mice, with MPO and CD68 staining intensity comparable to burn. 

Conclusion:  The alpha-7 nAChR is required for vagal-mediated protection against burn-induced acute lung injury.  Therapies that alter the inflammatory response after injury by either enhancing vagal activity or activating the alpha-7 nAChR may limit acute lung injury and improve outcomes in severely burned patients.

 

26.03 Accelerated Myocutaneous Revascularization Following Graded-Ischemia in db/db Mice

Posted on December 22, 2014

R. M. Clark1, B. Coffman1, D. A. Ramirez2, S. Godoy2, S. A. Meyers2, T. McGregor2, S. Krishna2, P. G. McGuire3, T. R. Howdieshell1  1University Of New Mexico HSC,Surgery,Albuquerque, NM, USA 2Skinfrared LLC,Albuquerque, NM, USA 3University Of New Mexico HSC,Cell Biology And Physiology,Albuquerque, NM, USA

Introduction: Murine models have provided valuable insight into the pathogenesis of both diabetes and chronic wounds.  The db/db mouse possesses a spontaneous mutation in the leptin receptor gene resulting in obesity, hyperglycemia, hyperinsulinemia, hypercholesterolemia, and insulin resistance.  This strain is characterized by impaired epithelialization of excisional wounds, with little known about wound neovascularization.

Methods: A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of C57BL6 (wild-type) and db/db mice (n=16 total; 5 mice per operative group and 3 unoperated mice per group as controls). Planimetric analysis of serial digital photographic images was utilized to determine flap viability in wild-type and db/db mice. Real-time myocutaneous flap perfusion and surface temperature were determined by laser speckle contrast and thermal infrared imaging respectively. Mice with surgical flaps were sacrificed on postoperative day 10. Image analysis of CD-31 immunostained sections confirmed flap microvascular density and anatomy. Quantitative RT-PCR was performed on nonoperative back skin and postoperative flap tissue specimens to determine local gene expression.

Results: Day 10 planimetric analysis revealed a mean flap viability of 95% in db/db mice compared to 79% in wild-type mice, and 60% of wild-type mice developed distal flap dehiscence not evident in db/db mice. Over 10 days, laser speckle contrast imaging documented markedly increased perfusion at all times points (p<0.001) with functional revascularization to supranormal perfusion in db/db flaps. In contrast, wild-type flaps displayed expected graded flap ischemia with failure to return to baseline perfusion during the postoperative period. Thermal infrared imaging documented complementary spatiotemporal assessment of thermal-metabolic tissue state. Immunostaining confirmed significant differences in preoperative and postoperative microvascular density (mean post-op distal vessel count 60 ± 8 versus 80 ± 15 vessels/mm2 for wild-type and db/db mice respectively, p=0.018; mean post-op distal vascular surface area 4108 ± 95 versus 8250 ± 795 µm2/mm2 for wild-type and db/db mice respectively, p<0.001). Finally, quantitative RT-PCR demonstrated statistically significant differences in angiogenic gene expression between wild-type and db/db mice at baseline (unoperated) and at day 10.

Conclusion: In a graded-ischemia wound healing model, accelerated myocutaneous revascularization and improved wound healing were evident in db/db mice compared to wild-type controls. Gene expression analysis reveals the db/db mouse may be “primed” for wound neovascularization and warrants further investigation into the role of the leptin-leptin receptor axis in wound angiogenesis.

 

 

22.10 Acid Sphingomyelinase Inhibition Decreases Lung Injury after Transfusion with Stored Blood

Posted on December 22, 2014

R. S. Hoehn1, P. L. Jernigan1, E. F. Midura1, J. M. Sutton1, C. C. Caldwell1, M. J. Edwards1, E. Gulbins1,2, T. A. Pritts1  1University Of Cincinnati,Surgery,Cincinnati, OH, USA 2University Of Duisburn-Essen,Molecular Biology,Essen, ESSEN, Germany

Introduction:  Transfusion of human packed red blood cell units (pRBCs) is ideal for resuscitation after hemorrhage. However, studies have shown that use of aged pRBCs are associated with increased morbidity and mortality compared to fresh units. Our laboratory and others have demonstrated that pRBC storage results in accumulation of microparticles that cause increased lung inflammation after transfusion. Ceramide, a sphingolipid formed by the enzyme acid sphingomyelinase (Asm), is present in cell membranes and mediates a variety of cell signaling and membrane changes in red blood cells, but its role in microparticle formation is unknown. We hypothesized that Asm inhibition would lead to decreased ceramide accumulation and microparticle formation in pRBCs, with a resultant decrease in lung injury after hemorrhage and resuscitation.

Methods:  Human and murine pRBCs were obtained and treated with increasing concentrations of Amitriptyline (AT), a specific Asm inhibitor, or vehicle, then stored under standard blood banking conditions. At intervals, microparticles were isolated from pRBC units and quantified with Nanoparticle Tracking Analysis. Asm activity and ceramide concentration were measured in stored erythrocytes and microparticles. Mice underwent hemorrhage and resuscitation with equal numbers of microparticles from AT- and vehicle-treated pRBC units. Lungs were collected from shocked mice and assessed for inflammatory cytokines and histology.

Results: During storage, pRBC units demonstrated significantly increased microparticle formation, ceramide accumulation, and Asm activity. Human and murine pRBCs treated with AT formed fewer microparticles in a dose-dependent manner (Figure 1A). AT-treated pRBCs and microparticles contained less ceramide and Asm activity. After hemorrhage, mice resuscitated with microparticles isolated from vehicle-treated pRBCs had significantly elevated lung levels of pro-inflammatory cytokines including macrophage-derived chemokine (Figure 1B), IL-1b, IL-6, KC, MCP-1, MIP-1a, and MIP-2 as well as increased lung damage as determined by histology. Resuscitation with equal numbers of microparticles from AT-treated pRBCs resulted in significantly decreased lung injury.

Conclusion: Amitriptyline treatment of pRBC units leads to decreased Asm activity, ceramide formation, and microparticle accumulation. Asm inhibition caused alterations in the microparticles that led to decreased acute lung injury following hemorrhage and resuscitation. Asm inhibition represents a novel opportunity to mitigate harmful effects of resuscitation with stored pRBCs.

 

« Previous Page
Next Page »