63.10 Progenitor Rescue Model for the Investigation of Vascular Restenosis

Posted on December 22, 2014

B. W. Tillman1, T. D. Richards1, V. S. Donnenberg2  1University Of Pittsburgh Medical Center,Division Of Vascular/Department Of Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh Medical Center,Department Of Cardiothoracic Surgery,Pittsburgh, PA, USA

Introduction: Restenosis threatens the longevity of over half of all vascular interventions and circulating progenitor cells have been implicated for their role in this pathology.  This contrasts to the conventional paradigm of smooth muscle proliferation in restenosis.   Our laboratory has previously demonstrated significant reductions in restenosis after depletion of circulating progenitor cells.  Vascular injury signals are also believed to represent an important initial step in restenosis.  The exact role of vascular injury signals in restenosis, however, has been difficult to examine given overlapping roles in both progenitor mobilization and smooth muscle proliferation.  Previous reports have suggested reduced restenosis among mice with knockout of the vascular injury signal, matrix metalloproteinase 9, (MMP9KO).   We hypothesized that this finding may be related to impaired progenitor surge.  We further postulated that an approach of administering stem cell factor (SCF) may rescue progenitor surge even in the setting of impaired vascular injury signaling.

Methods: Transgenic MMPKO mice underwent wire injury of the femoral artery as part of an IACUC approved study. Blood was examined on post-operative day 1 using flow cytometry for CD34+/c-kit+ progenitor cells and results were compared to non-operative controls.  In a separate experiment, MMP9KO mice were treated with SCF for 4 days prior to flow cytometric analysis of blood specimens.   Significance of the results was determined using the Student’s t-test.  

Results: In contrast to our findings of progenitor surge after arterial wire injury in C57BL6 animals, our results in MMP9KO mice reveal no significant change among progenitors after wire injury compared to non-operative controls (P=0.74).   Administration of SCF demonstrated an average 2.3 fold increase among progenitors compared to saline treated controls (P=0.007)

Conclusion: In summary, our findings suggest that the vascular injury signal MMP9 is an essential factor in progenitor surge after vascular injury.  Increased progenitors after administration of SCF suggest this cytokine rescues progenitor surge even amidst deficiency of upstream MMP9.   This model presents a unique opportunity to examine the effects of progenitor cells on restenosis independent of the impact of vascular injury signals on vascular smooth muscle proliferation.  These results further support a model of vascular injury induced progenitor mobilization that ultimately contributes to restenotic pathology.

 

63.03 Mechanisms of Erythropoietin-Mediated Neuroprotection Following Spinal Cord Ischemia Reperfusion

Posted on December 22, 2014

L. S. Foley1, J. Mares1, F. Puskas1, M. T. Bell1, D. T. Bennett1, K. Freeman1, M. Weyant1, D. A. Fullerton1, T. B. Reece1  1University Of Colorado Denver,Surgery/Cardiothoracic,Aurora, CO, USA

Introduction:  Delayed paraplegia is a devastating complication of thoracoabdominal aortic surgery.  Erythropoietin (EPO) attenuates this injury in models of spinal cord ischemia. Proposed mechanisms include induction of ischemic tolerance in normal tissue and activation of an ischemia-induced receptor, which attenuates neuronal loss in ischemic tissue. This study aims to elucidate mechanisms of EPO-mediated neuroprotection.  We hypothesized that EPO acts on ischemic tissue to limit neuronal loss.  

Methods:  Adult male C57/BL6 mice underwent sternotomy and 4-minute thoracic aortic crossclamp. Seven pretreatment mice received EPO (20U/kg) via intraperitoneal injection 24 hours prior to operation. Seven perioperative mice received the same EPO dose 4 hours prior to operation. Seven controls received 0.9% saline.  Four sham mice underwent saline administration and sternotomy without crossclamp. Functional outcomes were measured using Basso motor score for 48 hours (Basso score ranges 1-9; 1 refers to complete paralysis and 9 is normal hind-limb motor function).  Spinal cords were harvested and homogenized for biochemical analysis. 

Results:  Ischemia reperfusion (IR) injury uniformly induced paraplegia in control mice (p<0.01).  Synchronous EPO administration with IR prevented motor decline, which occurred in ischemic controls and EPO pretreated mice starting at 36 hours post-op (mean Basso 6.43 vs 1.86 and 3, respectively; p<0.01).  All ischemic control and pretreatment mice progressed to paraplegia, with no significant difference in hind limb function at all time points.

Conclusion:  EPO administration attenuates functional decline following spinal cord ischemia reperfusion injury when administered concomitant to the injury, but this effect is lost when EPO is given as a pretreatment.  These findings point towards a potential salvaging role for EPO following neurologic injury. Elucidation of mechanisms involved in spinal cord protection is essential for reducing delayed paraplegia. 

 

62.11 Current and Future Approaches to the Proteomic Analysis of Traumatic Coagulopathy

Posted on December 22, 2014

C. C. McCoy1, E. Benrashid1, M. L. Shapiro1, S. N. Vaslef1, J. H. Lawson1  1Duke University Medical Center,Department Of Surgery,Durham, NC, USA

Introduction:
Current translational research in traumatic coagulopathy is limited by the heterogeneity of individual injuries and the acute, unpredictable nature of trauma incidence.  As a result, coagulation studies of plasma from trauma patients lack baseline comparisons and are biased by numerous clinical factors including multisystem trauma, pre-hospital resuscitation and blood component administration.  Developing both animal and human models of trauma will permit a more rigorous characterization of coagulation changes resulting from specific organ injury.

Methods:
A mouse model of blunt traumatic brain injury (TBI) was utilized to create a pilot study of TBI-specific coagulation changes.  Three mice received piston-based, blunt TBI (6.8 m/s, 3mm deflection) following anesthesia and scalp retraction, while three mice received anesthesia and scalp retraction alone.  Thirty minutes following intervention, blood was obtained and a proteomic analysis of coagulation was performed.  2-dimensional difference gel electrophoresis (DIGE) was coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify proteins with altered concentration in an unbiased fashion.

Results:
Multiple coagulation-related proteins were detected by DIGE, and three known coagulation proteins demonstrated concentration alterations (greater than 5% concentration change) following trauma.  Five novel proteins of unknown function also demonstrated alterations in concentration.  Although previously uncharacterized, these proteins could serve as targets for future investigation of injury-induced changes in the plasma proteome.

Conclusion:
Detecting concentration changes of known coagulation factors and novel proteins during TBI reinforces the value of organ-specific models of trauma as a mechanism to study the plasma proteome during injury.  To validate animal model findings, proteomic analyses will be performed on human plasma collected before, during and after elective surgery to develop human, organ-specific injury models.  Data from such research will enhance our knowledge of coagulation changes during tissue trauma, the influence of inflammatory pathways, and their relationship to organ-specific injury.

62.10 Donor Plasma Effects On Platelet Function

Posted on December 22, 2014

A. G. Grand1, J. C. Cardenas1, L. Baer1, N. Matijevic1, B. A. Cotton1, J. B. Holcomb1, C. E. Wade1  1University Of Texas Health Science Center At Houston,Department Of Surgery,Houston, TX, USA

Introduction: We and others have demonstrated a significant decrease in platelet count and function within 2 to 3 hours following admission in severely injured trauma patients after transfusion. The decrease in platelet function is in part due to the reduction in platelet count. However, the decrease in function does not always equal the decrease in count observed in trauma patients. We investigated whether transfusion of plasma was another factor contributing to platelet hypofunction following trauma.

Methods: Whole blood samples were taken from healthy volunteers and their baseline platelet function assessed by impedance aggregometry in response to ADP, collagen, thrombin receptor-activating peptide (TRAP), arachidonic acid (AA) and ristocetin using Multiplate Analyzer. Blood samples were then diluted by 30% using the volunteer’s autologous plasma, autologous plasma that was snap frozen and thawed (autologous FFP), and donor fresh frozen plasma (FFP) from Gulf Coast Regional Blood Center. The percent change in platelet function compared to whole blood or autologous FFP was calculated. FFP from five different donors were used to obtain the average change in function. A student’s t test with significance set at p<0.05 was used to determine if dilution, freezing and the use of donor FFP had an effect on platelet function.

Results: Dilution of whole blood with autologous plasma by 30% showed a significant decrease of 30% in platelet function in response to all agonists, as expected, with the exception of TRAP. Autologous FFP had no additional effect on platelet function, with the exception of a reduction in TRAP (p=0.007). Single donor plasma demonstrated a further reduction in ADP (p=0.02), collagen (p=0.006) and AA (p=0.007) compared to autologous FFP. Finally, comparison across multiple donors (n=5) demonstrated a trending, although not significant, reduction in platelet function in response to all agonists with the exception of ristocetin, which remained unchanged.

Conclusion: Dilution is the major contributor in the decrease in platelet function however, donor plasma may have additional negative effects on platelet function following transfusion.  

 

62.09 Angiotensin Receptors In The Small Bowel Of Rats: A Novel Target For Hypertension

Posted on December 22, 2014

R. Patron-Lozano2, M. I. Rodriguez-Davalos1, A. Munoz-Abraham1, J. P. Geibel1,2  1Yale University School Of Medicine,Transplantation/Surgery,New Haven, CT, USA 2Yale University School Of Medicine,Surgery,New Haven, CT, USA

Introduction:  The renin-angiotensin-aldosterone system, through angiotensin II (AII), is one of the most important blood pressure regulators. The vasoconstriction caused by AII and the sodium balance effected by aldosterone have been a key target for drugs aiming to control hypertension. Angiotensin (AT) receptors have been well identified in vascular smooth muscle and renal tissue. Moreover, they have also been identified throughout the enteric epithelium, although mainly as a mitogenic agent. In the colon, the localization of functional angiotensin receptors has recently been described by our group and shows a promising potential future target for antihypertensive agents. In this study, we aimed to identify the presence of functional angiotensin receptors using intact small bowel of rats to determine if AII and AT1 receptor agents can modulate small bowel salt transport.

Materials and

Methods:  We used Sprague Dawley rats (280 and 360 grams) fasted for 18 hours, in order to reduce intestinal residue. The rats were anesthetized with isoflurane and euthanized. We took 12cm of distal ileum and stripped it from its mesentery. The intestinal lumen was flushed with lactated ringer’s solution in order to remove any remaining intestinal contents. The intestinal loop was then attached to a custom perfusion chamber where it was filled with 2mL of angiotensin at a concentration of 1×10-11 M and weighed. This measurement was determined as weight at t0. The chamber was then filled with lactated ringer’s at 37C and connected to a roller pump which continuously exchanged the fluid and maintained constant temperature for 1 hour. Then, the chamber was emptied and weighed again; this measurement being t1. For control purposes, the experiment was run using the same technique but the intestinal lumen filled with 2mL of lactated ringer’s. 

Results: We observed a significant decrease in weight after the administration of angiotensin at 10-11 M concentration between t0 and t1. This difference was consistent in every experiment. The mean weight difference was 93mg or 3.20% reduction in net graft weight. For the control samples, the weight remained the same at t1 or even slightly increased by almost 2% compared to the initial tissue weight.

Conclusion: We conclude that there are functional angiotensin receptors in rat small bowel that are sensitive to low dose AII(10-11M). By recording a difference in graft weight after the administration of low dose AII we proved that these receptors are not only present, but they can be stimulated provided adequate characteristics. These findings can further help us comprehend the extent of the fluid and electrolyte regulators in the body thus providing novel targets for control of hypertension and other diseases. 

 

62.08 Warm Ischemia-Reperfusion Injury Causes Epithelial to Mesenchymal Transformation in Human Cholangiocytes

Posted on December 22, 2014

J. Zhang1, N. Singh1, W. A. Dorsett-Martin1, C. D. Anderson1, T. M. Earl1  1University Of Mississippi,Division Of Transplant And Hepatobiliary Surgery, Department Of Surgery,Jackson, MS, USA

Introduction: The discrepancy between need and supply of liver allografts has led to alternative strategies to increase the organ pool.  One strategy, utilization of donation after cardiac death (DCD) donors, is severely limited by intolerance of the biliary system to warm ischemia resulting in ischemic cholangiopathy which reduces longevity of these allografts.  We hypothesize that warm ischemia followed by cold storage and subsequent reperfusion injury results in epithelial to mesenchymal transformation (EMT) and increased extra-cellular matrix production in human cholangiocytes.

Methods: Primary human cholangiocytes were exposed to no treatment (NT), hypoxic storage at 4°C in Histidine-tryptophan-ketoglutarate (HTK) solution for 7 hours (CIR group) or 3 hours hypoxic normothermic storage in PBS followed by 4 hours hypoxic storage in HTK at 4°C (WIR group).  Following 7 hours hypoxic storage (CIR and WIR), cholangiocytes were reperfused with media under normoxic, normothermic conditions.  Cells were harvested at 24, 72 and 144 hours post-reperfusion.  Quantitative real-time PCR for cholangiocyte epithelial marker AQP1, mesenchymal cell markers αSMA and S100α4, and COLA1 was performed at each time point and normalized to house-keeping gene 18S.

Results: One hundred forty-four hours after reperfusion, AQP1 expression is significantly diminished for WIR (WIR 6.2±0.3 vs. NT 210.6±33.8; p<0.001) but not after CIR (CIR 182.4±28.7 vs. NT 210.6±33.8 vs.; p=0.33) and was significantly lower in WIR compared to CIR (WIR 6.2±0.3 vs. CIR 182.4±28.7; p<0.001).  Interestingly, following CIR cholangiocytes lost Aqp1 mRNA at 24 and 72 hours but regained it by 144 hours.  Mesenchymal marker αSMA mRNA was significantly increased at 144 hours post-reperfusion in cholangiocytes exposed to WIR (NT 2.80±.3 vs. WIR 47.2±2.7; p<0.001), but decreased following CIR (NT 2.8±.3  vs. CIR 1.5±0.6;  p=0.02). This difference was not seen at 24 and 72 hours.  COL1A1 mRNA increased dramatically at 144 hours following WIR but not after CIR (WIR 130.0±6.0 vs. CIR 5.1±0.3; p<0.001), expression following CIR injury did not differ from NT group at any timepoint.  S100α4 trended toward significantly higher expression 144 hours following WIR (WIR 6.0±1.6, CIR 2.8±1.3; p=0.059).

Conclusion: Following warm-ischemia with subsequent cold storage and reperfusion injury human cholangiocytes lose epithelial cell markers, gain mesenchymal cell markers, and have increase expression of collagen 1 mRNA.  Further insight into this process may result in better utilization and success of DCD liver allografts. 

 

62.06 Relationship of Calcium Sensing and Angiotensin Receptors in the Small Bowel of Mice.

Posted on December 22, 2014

R. Patron-Lozano1, M. I. Rodriguez-Davalos1,2, A. Munoz-Abraham2, J. P. Geibel1,2  1Yale University School Of Medicine,Surgery,New Haven, CT, USA 2Yale University School Of Medicine,Transplantation/Surgery,New Haven, CT, USA

Introduction: The calcium sensing receptor (CaSR) has been identified and its functions described along the entire gastrointestinal tract. Recently, angiotensin receptors have been identified in the small bowel but their physiological function remains controversial. Since both systems are implicated in the regulation and modulation of transmembrane transport of fluids and electrolytes, and even inflammatory diseases, we chose to investigate if there was a connection between these two receptor classes. 

Materials &

Methods:  We used CaSR-/- and Gcmc2 double knockout mice and heterozygote (HET) mice for control. All mice were fasted for 18 hours to reduce intestinal residue; total body weight ranged between 23 and 50 grams. The mice were anesthetized with isoflurane and euthanized. We took 12cm of distal ileum and stripped it from its mesentery. The intestinal lumen was flushed with lactated ringer’s solution in order to remove any remaining intestinal contents. The intestinal loop was then attached to a custom perfusion chamber where it was filled with 2mL of angiotensin at a concentration of 1×10-11 M and weighed. This measurement was determined as weight at t0. The chamber was then filled with lactated ringer’s at 37C and connected to a roller pump which continuously exchanged the fluid and maintained a constant temperature. The experimental time period was 1 hour. The chamber was then emptied and weighed again; this measurement being t1. 

Results: For the knockout mice we observed a significant increase in weight after the administration of angiotensin at 10-11 M concentration between t0 and t1. This difference was consistent in every experiment. The mean weight difference was 73mg or a 3.14% increment in graft weight. For the control HET samples, the weight diminished consistently at t1 96mg or 3.31% of the initial tissue weight.

Conclusion: After performing these experiments, we conclude that there is a functional relationship between angiotensin receptors and calcium sensing receptors in the small bowel of mice. By recording a difference in graft weight after the administration of angiotensin at a concentration of 10-11 M in CaSR -/-;Gcmc2-/- mice we observed that although the angiotensin receptors might be present and stimulated, the absence of CaSR prevents normal physiologic function. Furthermore, by conducting a control experiment with the HET mice, we demonstrated that functional calcium sensing receptors aid the normal physiology of fluid exchange in the small intestine in the presence of angiotensin. These findings can further help us comprehend the extent of the fluid and electrolyte regulators in the body thus providing novel targets for control of hypertension and other diseases.
 

62.05 Ex-vivo Normothermic Perfusion (EVNP) to Assess and Repair Kidney Ischemia-Reperfusion Injury

Posted on December 22, 2014

I. Palma1, J. Woloszyn1, R. Abbott1, Y. Smolin1, R. Ramsamooj2, N. Tran2, C. Santhanakrishnan1, R. V. Perez1  1University Of California – Davis,Surgery,Sacramento, CA, USA 2University Of California – Davis,Pathology And Laboratory Medicine,Sacramento, CA, USA

Introduction: Hypothermic storage of organs has been a gold standard for organ preservation in transplantation. However, recent studies have shown that EVNP has the potential to repair and assess the viability of marginal organs prior to transplantation by restoring normal metabolism. The optimal perfusion solution for these functions has not been determined.

Methods: Paired high risk human kidneys (n=8) deemed unsuitable for transplantation were placed on 3 hours of pressure dependent EVNP with a centrifugal pump at 37°C. Kidney was oxygenated via a hollow fiber membrane oxygenator and supplemented with parenteral nutrition & insulin. Kidneys were perfused with leukocyte depleted packed red blood cells (PRBC) as an optimal “repair” solution or whole blood (WB) to more accurately assess viability during early reperfusion.  Exogenous creatinine (0.06 g) was added to the system to assess glomerular clearance.  Pump parameters and urine output were monitored.  Blood and urine samples were collected at the start and every 30 minutes and analyzed for pH, oxygen, electrolytes, creatinine, lactate and neutrophil gelatinase associated lipocalin (NGAL) using a Triage point of care machine. NGAL is an acute kidney injury biomarker. Results were compared between groups using a general linear model for repeated measures test.

Results:The mean age of donors was 60 years. The mean static cold ischemia time was 52.5 hours. The Maryland Aggregate Pathology Index and Kidney Donor Prognostic Index were equal in both groups (5.25 and 87% respectively). Hemodynamic parameters evaluated were flow and resistance, and neither showed statistical significance (p=0.19 and p=0.20). However, PRBC showed a better trend in both parameters. Functional parameters appeared more favorable in the PRBC group but only achieved statistical significance with urine NGAL as shown in Table 1. 

Conclusion:EVNP of high-risk kidneys is possible and combined with POC testing offer a means of assessing organs in real time, potentially making it possible to transplant organs that previously were thought to be untransplantable.  Perfusion with PRBC may have a beneficial effect on the kidney, but may not accurately assess the degree of ischemia-reperfusion injury when compared to the more physiologic WB perfusion. More studies are warranted to determine the contributing factors and clinical usefulness of urinary biomarkers such as NGAL during EVNP.
 

62.04 Carnosol and Derivatives have Potential as Novel Organ Protective Agents

Posted on December 22, 2014

T. KAWAMURA1,2, T. MOMOZANE1,2, S. FUNAKI1, Y. SHINTANI1, M. INOUE1, M. MINAMI1, K. SUGIMURA2, O. IIDA2, H. FUCHINO2, N. KAWAHARA2, H. TAKEMORI2, M. OKUMURA1  1Osaka University Graduate School Of Medicine,General Thoracic Surgery,Suita, OSAKA, Japan 2National Institute Of Biomedical Innovation,Ibaraki, OSAKA, Japan

Introduction:  Oxidative stress is thought to be involved in various disease states related to organ transplantation such as ischemia/reperfusion injury and its abrogation in affected patients is critical. Carnosol is a plant-derived phenol that functions as an antioxidant and its mechanism of action involves activation of nuclear factor E2 p45-related factor 2 (Nrf2), which plays a key role in regulation of genes driven by antioxidant response element (ARE). Although it is considered to have potential for treatment of oxidative stress, disadvantages include instability in water and high cost, making it difficult to employ in clinical practice. In the present study, we investigated the lung protective effects of carnosol, extracted from Callicarpa longissima, and its derivatives in vivo using a warm lung ischemia model and in vitro with a lung cell line.

Methods:  C57BL/6J mice received 0.01% carnosol chow for 2 weeks, after which the left lungs were excised and cultured in DMEM at 37˚C. The concentration of lactate dehydrogenase (LDH) in medium was measured as a marker of lung damage. Following pre-treatment with carnosol, we assessed the expression of heme oxygenase (HO)-1 and H2O2 treatment tolerance in a lung cell line (NCI-H1975), and also evaluated activation of Nrf2 using a cell-based ARE-luciferase reporter assay. Furthermore, we investigated the stability of carnosol derivatives (methylated-, acetylated-, propionylated-carnosol) in a water solution.

Results: In the excised ischemic lungs, carnosol was shown to increase the amount of HO-1 protein and delayed the leakage of LDH (Fig. A). In NCI-H1975 cell cultures, pretreatment with carnosol induced HO-1, increased the amount of intracellular Nrf2, and protected against H2O2 treatment (Fig. B). Carnosol also increased ARE-luciferase activity in a dose-dependent manner. In addition, our results showed that carnosol induced HO-1 to a much greater degree than the same concentration of tBHQ, a representative Nrf2 inducer (Fig. C). Together, these findings suggest that carnosol induces HO-1 via multiple signaling pathways including Nrf2. Furthermore, carnosol derivatives were stable in a water solution and induced HO-1 up to 1 week after preparation.

Conclusion: Although additional investigation is required, carnosol and its derivatives can significantly reduce organ and cell damage by inducing HO-1, and may have potential as novel organ protective agents.

 

62.02 A new method to measure intestinal secretion using FITC-Inulin in small bowel of rats

Posted on December 22, 2014

A. Munoz-Abraham1, G. Torres-Valencia1, T. Alfadda1, C. Jasinski1, R. Patron-Lozano1, M. I. Rodriguez-Davalos1, J. P. Geibel1  1Yale University School Of Medicine,Surgery – Transplant,New Haven, CT, USA

Introduction: Intestinal ischemia remains a major limitation in successful intestinal transplantation. Several animal intestinal ischemia models have been developed and used. However, rats remain as the most commonly used. The fragile balance of the intestinal mucosa relies on stable homeostatic mechanisms. To further understand the disruption of these mechanisms during ischemia/reperfusion injury, we have developed a new method to objectively measure osmotic changes in the intestinal lumen of rats that equate to injury.

Material and

Methods:  We used Sprague Dawley rats (401 to 442 grams).The rats were anesthetized and euthanized with isofluorane. 20 cm of distal ileum were taken and stripped from the mesentery. The intestinal lumen was flushed with regular HEPES solution (pH 7.408, mosm 297) to remove any remaining intestinal debris . The intestinal loops were then attached to two custom perfusion chambers that received a constant flow of regular HEPES solution. The chambers were submerged in a bath of deionized water at 37 C . At time 0, a known concentration of 3 ml of 0.001 mM FITC-Inulin was perfused into both lumens. Samples were collected and Relative Fluorescence Units (RFU) were measured using the NanoDrop 3300 Fluorospectrometer. The control intestine remained receiving a flow of regular HEPES, while the experimental intestine was bathed with a flowing solution of 10 µM Forskolin in a perfusion volume of 200 ml regular HEPES. Samples from the lumen were again collected from both intestines at times 25 and 35 minutes, and measured.

Results: A significant increase in luminal secretion was observed after the administration of the 10 µM Forskolin solution by observing the decrease in RFU units by almost half in the experimental intestine compared to the control.

Conclusion: It can be concluded that the use of FITC-Inulin can be an effective and objective method to measure fluid secretion or absorption in the small bowel, thus giving a more accurate estimate of the viability of the organ. By measuring the difference in RFU of a known solution after stop flow in the small bowel we demonstrated that absorption and secretion processes take place depending on the pathophysiological state of the organ. Absorption was observed when the intestine was maintained in physiological-like conditions, while secretion of large amounts of water into the lumen occurred when FSK was administered. In both cases the change in RFU using the method proposed gave accurate reproducible results with small sample variations  demonstrating the usesfulness of this model system for assessing intestinal viability.

 

62.03 Green Plasma Has a Superior Hemostatic Profile Compared with Standard Color Plasma

Posted on December 22, 2014

B. A. Cotton1, J. C. Cardenas1, E. Hartwell1, C. E. Wade1, J. B. Holcomb1, N. Matijevic1  1University Of Texas Health Science Center At Houston,Acute Care Surgery/Surgery,Houston, TX, USA

Introduction:  While the transfusion of plasma has increased over the last decade, the

availability of this product has seen dramatic changes that continue to threaten the current

supply. The conversion to male predominant plasma has further limited potential donors

of emergency release products used for intial resuscitaion in trauma. During this same

period, investigators have demonstrated a sexual dimorphism in response to sepsis and

injury, with less multiple organ failure and improved survival observed in premenopausal

females. Plasma from female donors who are pregnant or taking oral contraceptives often

has a green appearance. This green discoloration (due to increased ceruloplasmin levels)

frequently results in these units being discarded or removed from the donor pool for

commercial use, purely based on its appearnace. The purpose of this pilot study was to

evaluate the hemostatic potential and capacity of green plasma compared to standard

color plasma.

Methods:  We obtained plasma from 12 blood group-matched female donors from our

local blood center. Six of these units had a normal appearing hue (STANDARD) and six

were grossly green-appearing plasma (GREEN). These units were then evaluated by

thrombelastography (TEG), calibrated automated thrombogram (CAT) and factor level

measurements. Univariate analysis was then performed using Wilcoxon rank sum and

values are expressed in medians with 25th and 75th interquartile range.

Results: GREEN plasma had a more hypercoagulable TEG profile for all values (r-value:

1.6 vs. 3.1 min, p=0.004; k-time: 2.2 vs. 4.8 min, p=0.088, angle: 69 vs 42 degrees,

p=0.004; mA: 38 vs. 25 mm, p=0.054) when compared to STANDARD plasma.

Differences were also observed with coagulation factor levels comparison, with

GREEN plasma having higher levels than STANDARD (factor II: 107 vs. 96%, p=0.004;

factor VII: 124 vs. 106%, p=0.149; factor IX: 145 vs. 114%, p=0.077; factor X: 125 vs.

102%, p=0.006; factor XI: 121 vs. 101%, p=0.025). Using CAT, GREEN plasma had

higher lag time (4.1 vs. 3.6 min, p=0.037) and increased endogenous thrombin potential

(1669 vs. 1280 nM/min, p=0.114).

Conclusion: This pilot study demonstrates that plasma from female donors that has a green color has a

superior hemostatic profile than standard color plasma. Current AABB recommendations

for male-predominant plasma have further reduced the availability of emergency release

plasma for life-threatening bleeding. GREEN plasma should be further investigated for

its safety profile and hemostatic potential. Should it prove to be a safe and functionally

non-inferior (and potentially superior) product, GREEN plasma should be actively

re-introduced into the medical community for transfusion of critically injured and

bleeding patients.

61.10 Differences in Treatment-Induced Perfusion Following Flap Surgery

Posted on December 22, 2014

T. Ramesh1, N. Patel1, G. Aaron1, J. Warram1, E. Rosenthal1  1University Of Alabama At Birmingham,Department Of Surgery,Birmingham, AL, USA

Introduction:  Minimizing surgical morbidity after local flap reconstruction is important in the management of cutaneous defects. In the past, many treatment modalities have been used to mitigate the disease burden and preserve the function of vital head and neck structures. Controversy exists in the literature regarding the effects of two such modalities—radiation and chemotherapy—on flap perfusion. Neoadjuvant treatments have the potential to cause damage to the microvasculature of the surgical bed through fibrosis, endothelial cell damage, and reduced cell proliferation, all of which increase the likelihood of postoperative flap failure. The aim of the present study is to examine the effects of neoadjuvant radiation and chemotherapy on blood perfusion following dorsal flap surgery in an athymic female mouse model. 

Methods:  Animals were divided into three treatment groups: No pre-flap treatment (negative control, n=4), 36-Gy electron-beam radiation administered to dorsal skin (radiation group, n=4), and 2 mg/kg intraperitoneal cisplatin (chemotherapy group, n=4). Treatments were completed on mice 15 days prior to undergoing random-pattern dorsal flap surgery with a length-to-width ratio of 4:1 (4x1cm2). Flap perfusion was assessed via laser-assisted indocyanine green dye angiography and by standard clinical assessment.

Results: LUNA perfusion imaging performed on post-operative day 1 showed 56% distal end flap perfusion relative to healthy skin in chemotherapy group mice, compared to 69% and 71% distal end perfusion in control and radiation groups, respectively. LUNA perfusion imaging performed on post-operative day 4 showed 75% and 72% perfusion relative to healthy skin in control and radiation group flaps, respectively. By post-operative day 5, all chemotherapy group flaps experienced full flap loss. In contrast, 3 of 4 control group flaps and all radiation group flaps survived to the conclusion of the experiment. Clinical assessment of flap perfusion by two physicians on post-operative day 2 revealed that elevated skin flaps of the chemotherapy group were more poorly perfused throughout than flaps in control and radiation groups.

Conclusion: Both LUNA intraoperative imaging and clinical judgment indicated that distal ends of chemotherapy group flaps were most poorly perfused. In addition, complete flap loss occurred faster in chemotherapy group mice than in radiation group and control group mice, suggesting that chemotherapy treatment has the most detrimental effect on flap viability.

 

60.13 Feasibility of Arteriovenous Fistula Creation After Previous Radial Artery Harvesting for Aortocoronary Bypass

Posted on December 22, 2014

C. Rivera1, N. J. Gargiulo1  1North Shore University And Long Island Jewish Medical Center,Manhasset, NY, USA

Introduction:   Arteriovenous fistula (AVF) formation remains the procedure of choice in patients requring hemodialysis. The feasibility of AVF creation in the setting of prior radial artery harvesting after aortocoronary bypass remains unknown. This investigation elucidates which patients might be candidates for AVF creation despite prior radial artery harvesting.

Methods:   A retrospective review was performed on 2,100 patients undergoing hemodialysis access procedures from 2003 to 2010. Of these patients, 11 (0.5%) were identified as having prior radial artery harvesting for aortocoronary bypass. Pre/Post-operative vein mapping, arterial duplex, digital plethysmography, selective angiography, and sestamibi scanning was performed to evaluate the ulnary artery and palmar arch. Patients with evidence suggesting an intact ulnar artery circulation then underwent AVF creation.

Results:  All 11 patients had an adequate preoperative work up.  Seven (64%) of the 11 patients had digital plethysmography suggesting an intact ulnar artery/palmar arch and underwent successful AVF creation.  Three (27%) of the patients had a variety of findings precluding successful AVF creation.  One (9%) patient with normal preoperative plethysmography developed a steal syndrome requiring revision of the arteriovenous fistula.

Conclusion:  Successful AVF creation is feasible in patients with prior radial artery harvesting for aortocoronary bypass. The use of preoperative digital plethysmography, selective ulnar artery/palmar arch arteriography and sestamibi scanning to evaluate forearm muscle perfusion may be used as adjuncts to guide a successful intervention.

 

43.09 Effect of Limb Demand Ischemia on Autophagy and Mitochondrial Biogenesis in Diet Induced Obese Mice

Posted on December 22, 2014

H. Albadawi1,3, R. Oklu2,3, J. D. Milner1, T. P. Uong1, H. Yoo1, M. T. Watkins1,3  1Massachusetts General Hospital,Department Of Surgery, Division Of Vascular And Endovascular Surgery,Boston, MA, USA 2Massachusetts General Hospital,Department Of Imaging, Division Of Interventional Radiology,Boston, MA, USA 3Harvard School Of Medicine,Brookline, MA, USA

Introduction: Diabetes is a major risk factor for peripheral arterial disease, which frequently manifests as intermittent claudication due to lower limb demand ischemia (DI). In skeletal muscle, autophagy and mitochondrial biogenesis are known to mediate oxidative capacity and energy metabolism. In aging and diabetes, defective mitochondrial biogenesis, including mitochondrial fission/fusion events and autophagy, contributes to structural and metabolic derangements in muscle. This study evaluates the effect of exercise and DI on autophagy and mitochondrial biogenesis in a diabetic mouse model of diet induced obesity.

Methods: Two groups of C57BL6 male mice were fed a 60% high fat diet for 26 weeks to induce diabetes. Mice were subjected to unilateral femoral artery ligation (FAL, n=6) or sham surgery (n=6) and were allowed to recover for 14 days for stabilization. Subsequently, the FAL mice underwent 60 minutes of daily treadmill exercise (12m/min speed, 10° incline) for 4 weeks to induce DI in the ligated limb. The exercised contralateral (EX) limb acted as an internal control to assess the effect of exercise on muscle independent of DI. In contrast, sham (SH) mice remained sedentary. Hind limb perfusion was recorded at baseline before surgery and once a week thereafter. Muscle tissues from SH, DI, and EX hind limbs were analyzed by western blotting for markers of autophagy, LC3B type II/I ratio, and markers of mitochondrial biogenesis, which was quantified by the expression ratio of mitochondrial fusion (Opa-1) and fission (Drp-1) proteins. Specific band density ratios were calculated and expressed in arbitrary units (AU). Statistical analysis was performed using ANOVA with post hoc analysis.

Results: After 4 weeks of sedentary or exercise conditions, EX perfusion was significantly higher than in SH or DI (SH: 1249±100; EX: 2032± 307; DI: 998±207 Flux, p<0.01). There was significant enhancement in LC3BII/ I ratio in the EX muscle over SH (p<0.01), while DI exhibited an insignificant increase over SH (SH: 0.68±0.06; EX: 2±0.4; DI: 1.2±0.3 AU). In the DI muscle, we established a significant positive linear regression between limb perfusion and the LC3B II/I ratio (r2=0.81, p=0.014). In contrast, comparison of the Opa-1/Drp-1 ratio failed to yield significance between the three groups (SH: 1.1±0.3; EX: 2±0.7; DI: 1.8±0.5 AU, p=0.332).

Conclusion: Exercise upregulated autophagy in the tissue with normal limb perfusion, but it failed to enhance autophagy in the limb that experienced demand ischemia. Variable levels of autophagy were observed to positively correlate with limb perfusion in demand ischemia. Furthermore, exercise does not appear to significantly alter mitochondrial biogenesis in diabetic muscle regardless of the degree of perfusion. These results may have an implication for the evaluation of diabetic patients, who suffer from claudication.

43.01 Sirt1 Stimulation Preserves Mitochondria and Enhances Autophagy in Hepatocytes after Hypoxic Injury

Posted on December 22, 2014

A. Khader1,2, W. Yang1,2, J. M. Prince1, J. Nicastro1, G. F. Coppa1, P. Wang1,2  1Hofstra North Shore-LIJ School Of Medicine,Surgery,Manhasset, NEW YORK, USA 2Elmezzi Graduate School Of Molecular Medicine,Manhasset, NEW YORK, USA

Introduction: Liver ischemia-reperfusion (I/R) often occurs in trauma, transplantation and prolonged shock state, where it lacks specific treatment. We have recently demonstrated that pharmacologic sirtuin 1 (Sirt1) activation, with SRT1720, is protective in a murine model of liver I/R. However, the detailed mechanism of which is not yet characterized. Sirt1 is an energy-sensing enzyme with multiple roles, including the regulation of energy metabolism, mitochondrial function and autophagy. We therefore hypothesized that SRT1720 protects hepatocytes from hypoxia-induced injury through the activation of mitochondrial biogenesis and the autophagy salvage pathway.

Methods:  Rat hepatocyte epithelial H4IIE cells were subjected to 6 h of hypoxia using 5% Oxyrase, followed by reoxygenation in the presence or absence of SRT1720 (500 nM) for 4 or 24 h. Cell survival was determined by cell counts and ATP assay. Cells were stained with MitoTracker green and red FM for measuring mitochondrial mass (mtMass) and membrane potential (MMP), respectively, by flow cytometry. Acridine orange stain was used to assess the formation of autophagic vesicles under fluorescent microscopy. Protein levels were determined by Western blot. 

Results: We observed a 27.6% and 21.7% reduction in cell numbers and ATP levels, respectively, at 4 h of reoxygenation (H/R), while SRT1720-treated cells were comparable to the normoxia control. At 4 h of H/R, the mtMass and MMP per cell, as measured by mean fluorescent intensity (MFI), were decreased by 44.6% and 27.6%, respectively, compared to the normoxia control. In contrast, treatment with SRT1720 enhanced both parameters by 3.4- and 5.5-fold, respectively, compared to untreated cells at 4 h of H/R. This enhancement of mtMass and MMP by SRT1720 persisted to 24 h of reoxygenation. Treatment with SRT1720 increased the expression of PGC1α, the master regulator of mitochondrial biogenesis, by 11.0% and 36.7% at 4 h and 24 h of H/R, respectively. In addition, there was a significant enhancement in autophagy in the SRT1720 treated cells as demonstrated by an increase in LC3aII/LC3aI protein ratio to 3.3 compared to 1.7 in the untreated cells at 4 h of H/R. Further, there was an observable increase in autophagic vesicles (bright contrast) at 24 h of reoxygenation which was remarkably increased by SRT1720, as shown by the increase in vesicle numbers and intensity of acridine orange precipitation (Figure).

Conclusion: Pharmacologic activation of Sirt1 improves the survival of hepatocytes under hypoxic stress, which is associated with maintenance of mitochondrial mass and integrity as well as an increase in autophagic activity. Thus, stimulation of mitochondrial function may provide a new protective strategy against liver I/R injury. 

43.02 Cytokine-induced MicroRNA-155 Limits iNOS Synthesis in Hepatocytes During Redox Stress

Posted on December 22, 2014

P. R. Varley1, Z. Guo1, D. A. Geller1  1University Of Pittsburgh,General Surgery,PITTSBURGH, PA, USA

Introduction:  Redox stress is an important mediator of liver damage ischemia reperfusion (I/R) injury. Previously our group has described the role of inducible nitric oxide synthase (iNOS) in the liver, which exacerbates liver injury under redox stress.  MicroRNA (miR)-155 is up-regulated by a number of inflammatory stimuli, however its role in the liver and effect on iNOS expression have not been reported. We hypothesized that miR-155 is induced in hepatocytes during inflammation and regulates iNOS expression.

Methods:  Rat and human hepatocytes were isolated through collagenase dissociation and density gradient centrifugation. To determine the ability of miR-155 to regulate iNOS expression, primary human hepatocytes were isolated and treated with miR-155 mimics and then stimulated with a cytokine mix (CM) ((interferon-γ, IL-1β and TNF-α) for 24 hours. The Griess method was used to measure nitrite concentrations in the cell culture supernatant as endproduct for induced NO synthesis. To evaluate miR-155 expression, rat hepatocytes were treated with hydrogen peroxide (H2O2) and CM alone or in combination for up to 24 hours. Trizol purification was used to isolate RNA from the cells, and miR-155 levels were measured with TaqMan qRT-PCR. 

Results: Transfection of primary human hepatocytes with miR-155 mimics resulted in near complete abrogation of nitrite production in CM-treated cells (NO2–  1.1 uM in miR-155+CM vs. 18.5 uM in CM alone, p < 0.05) suggesting significant suppression of cytokine-induced NO synthesis.  H2O2 stimulation alone had a non-significant effect on miR-155 expression at 24 hrs, while CM stimulation resulted in significant up-regulation of miR-155 expression at 24hr (Table 1). Co-stimulation of hepatocytes with CM + H2O2resulted in a dramatic 21.3-fold increase in miR-155 expression at 24 hours.

Conclusion: This study is the first to describe down-regulation of iNOS by miR-155, a well-known inflammatory microRNA. Not only is miR-155 capable of iNOS regulation, but here we show that inflammatory cytokine stimuli under redox stress act in a synergistic fashion to induce miR-155. Our previous work has shown the iNOS expression peaks at 6-12 hours after introduction of inflammatory stimuli and returns to baseline by 24 hours. Taken together with the data from this study, we suggest that miR-155 blocks iNOS-mediated NO synthesis in a post-translational manner as a homeostatic control mechanism to prevent the untoward consequences of excessive iNOS expression and limit hepatocellular damage.

 

43.03 Human Mesenchymal Stem Cells Improve Survival Following Intestinal Ischemic Injury

Posted on December 22, 2014

T. D. Crafts1, E. Blocher-Smith1, T. A. Markel1  1Indiana University School Of Medicine,Pediatric Surgery,Indianapolis, IN, USA

Introduction:   Cellular therapy is a novel surgical treatment option for intestinal ischemia.  Surgeons have direct access to injured bowel at the time of laparotomy and could use the intraperitoneal application of stem cells to salvage necrotic or marginal appearing intestine.  Bone Marrow Derived Mesenchymal Stem Cells (BMSCs) have previously been shown to abate the damage caused by intestinal ischemia/reperfusion injury (I/R)   We hypothesized that: 1) application of human BMSCs to intestine following I/R would improve survival, and 2) improved outcomes would be associated with increased tissue levels of angiogenic and intestinal growth factors, namely IL-6, VEGF, and CXCL-10.

Methods:   Adult BMSCs were cultured on polystyrene flasks in alpha-MEM containing fetal bovine serum (37C, 5%CO2 in air).  Cells were subcultured or used for experimentation at 90% confluence between passages 3-10.  Eight week old male C57Bl6J mice (20-25g) were then anesthetized and underwent a midline laparotomy.  I/R and MSC groups were exposed to superior mesenteric artery ligation for 60 minutes with a non-traumatic clamp.  Immediately following removal of the clamp, 2 x 106 human BMSCs in PBS were placed into the abdominal cavity.  Animals were then closed in two layers and allowed to reperfuse for 6 hours (molecular analysis) or 7 days (survival analysis).  Following 6 hour reperfusion, animals were euthanized.  Intestines were harvested and homogenized in RIPA buffer with phosphatase and protease inhibitors.  Extracts were quantified for total protein content (Bradford Assay) and analyzed by multiplex beaded assay for IL-6, VEGF, and CXCL10. P<0.05 was significant.

Results:  I/R caused marked intestinal ischemia and significant mortality.  Seven day survival was 30% for I/R, while application of MSCs following ischemia increased the seven day survival to 80% in a dose dependent fashion (p<0.05, Figure 1A).  MSC application increased intestinal tissue levels of IL-6 (IR: 39.6+/-5.2 ng/g protein, MSC: 84.3+/-19.2 ng/g protein, p=0.03) and CXCL10 (IR: 40.4+/-4.4 ng/g protein, MSC: 52.5+/-3.5 ng/g protein, p=0.03) but not VEGF (IR: 20.0+/-1.5 ng/g protein,  MSC: 22.4+/-3.0 ng/g protein, p=0.25)(Figure 1B-D).

Conclusion:  Direct application of human BMSCs to the peritoneal cavity following intestinal I/R improved survival by fifty percent.  Improved outcomes were associated with higher intestinal tissue levels of IL-6 and CXCL10, but not VEGF.  Further studies are needed to elucidate stem cell mechanisms in order to harness maximum therapeutic potential.

 

43.04 Anti-inflammatory effects of Carbon Monoxide are mediated through macrophages

Posted on December 22, 2014

A. E. Leake1,2, G. Hong1, B. S. Zuckerbraun3, E. Tzeng1,2  3University of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA 1VA Pittsburgh Healthcare System,Vascular Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh,Department Of Surgery, Division Of Vascular Surgery,Pittsburgh, PA, USA

Introduction:  Carbon Monoxide (CO) is a potent anti-inflammatory molecule and dramatically attenuates neointimal hyperplasia (NIH) after carotid injury with one hour of inhaled CO at 250 parts per million (PPM) just prior to injury.  The mechanism of how this brief treatment of inhalation of CO leads to profound and prolonged anti-inflammatory effects is still poorly understood.  We have shown marked and prolonged changes in macrophage phenotype following inhaled CO, and the responsiveness of these cells to inflammatory stimuli is significantly altered.  Based on these findings, we hypothesize that monocyte/macrophages mediate the vasoprotective effects of CO. 

Methods:  Sprague-Dawley rats (6-7/group) received Clodronate liposomes (5mg/mL IP) at day 0 for transient macrophage depletion and empty liposomes for control.  On day 3, rats were treated with inhaled CO (1-hr at 250ppm) or maintained in room air and then underwent left carotid artery balloon injury.  At the time of injury, a manual differential cell count of whole blood was used to confirm macrophage/monocyte depletion.  Two-weeks after injury, rats were sacrificed and carotid arteries were collected for histologic analysis for intimal hyperplasia (IH) by determination of intima/media ratio (I/M).  Statistical analysis was performed with ANOVA.

Results:IP administration of clodronate liposome resulted in a complete depletion of circulating monocytes by day 3 with a differential count of zero.  Control liposome treated rats receiving CO demonstrated a 50% reduction in IH compared to the air group (Figure; I/M = 0.38 ± .09 vs. 0.75 ± .08, respectively; P=.01).  After macrophage depletion, there was no difference in the amount of IH in the air treated rats compared to rats treated with control liposomes (I/M = 0.78 ± .09 vs. 0.75± .08, respectively; P=NS).  The protective effect of inhaled CO was completely reversed in the macrophage-depleted rats compared with the CO treated control rats (I/M = 0.83 ± .11 vs. 0.38 ± .09, respectively; P = .01).

Conclusion: CO significantly decreases IH formation in the rat carotid injury model.  This protective effect of CO is lost after macrophage depletion.  Macrophage depletion itself did not affect IH.  These findings support that the vasoprotective properties of inhaled CO are mediated through monocytes/macrophages.  This regulation of the innate immune system may explain the profound anti-inflammatory actions of inhaled CO and future studies will focus on deciphering the signaling pathways that link inhaled CO and the changes in macrophage phenotype.

43.05 PGC1α Helps Maintain the Intestinal Barrier and Protect Against Inflammation in Experimental Colitis

Posted on December 22, 2014

K. E. Cunningham1,2, G. Vincent1, D. J. Hackam1,2, K. P. Mollen1,2  1Children’s Hospital Of Pittsburgh Of UPMC,Pittsburgh, PA, USA 2University Of Pittsburgh,Pittsburgh, PA, USA

Introduction:  Defects in intestinal barrier function are known to be characteristic features of Inflammatory Bowel Disease (IBD).  Alterations in epithelial integrity are thought to predispose subjects to translocation of pathogenic bacteria and an activation of host defense mechanisms.  We recently demonstrated that Peroxisome Proliferator-activated Receptor γ  Coactivator 1α (PGC1α) is decreased in the intestinal epithelium of humans with severe IBD and mice undergoing experimental colitis.  Mice lacking PGC1α in the intestinal epithelium develop severe colitis due to a dramatic disruption of mitochondrial biogenesis and function.  We now hypothesize that PGC1α suppression leads to a breakdown of the intestinal barrier and an increase in bacterial translocation through alterations in mitochondrial dynamics.

Methods:  We created an intestinal epithelial-specific PGC1α knockout mouse, the PGC1αΔIEC mouse, by crossing a PGC1αflox/flox mouse with a villin-cre transgenic mouse.  PGC1αΔIEC mice and their wild-type littermates (PGC1αflox/flox) were subjected to DSS colitis for 7 days.  Animal weights and Disease Activity Index (DAI) were recorded daily.  Intestinal tissue was analyzed for expression of inflammatory cytokines, tight junction proteins, and barrier function.  H&E staining was used to evaluate inflammatory changes. Immunofluorescence was used to identify infiltrating bacteria within the intestinal mucosa as well as observe differences in the quantity and localization of tight junction proteins.

Results: Mice deficient in PGC1α within the intestinal epithelium demonstrate a significant decrease in the tight junction protein Occludin as demonstrated by western blot, PCR, and immunofluorescence during experimental colitis.  This leads to a dramatic increase in bacterial translocation and a worsening of intestinal inflammation.  Induction of PGC1α ameliorated experimental colitis.  

Conclusion: We demonstrate for the first time that the suppression of PGC1α that is seen within the intestinal epithelium of humans with severe, surgical IBD and mice undergoing experimental colitis leads to a disruption of the intestinal barrier and an increase in bacterial translocation.  Strategies aimed at enhancing PGC1α activity may improve treatment strategies for human IBD.

43.06 Oxidative Stress Induces Neutrophil Extracellular Traps in a TLR4- and PHOX-Dependent Mechanism

Posted on December 22, 2014

A. B. Al-Khafaji1, D. Miller2, H. Huang1, S. Tohme1, A. Tsung1  1University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA 2Beth Israel Deaconess Medical Center,Department Of Medicine,Boston, MA, USA

Introduction: Neutrophils accumulate in the liver after ischemia-reperfusion injury and contribute to inflammation-associated damage. Although intra-hepatocellular sources release reactive oxygen species after I/R, more significant to injury is neutrophils’ oxidative burst observed 6-24h after reperfusion. Neutrophils have recently been shown to extrude select intracellular contents to form a Neutrophil Extracellular Trap (NET). Stimulation of Toll-like receptors (TLRs) initiates a signaling cascade that includes activation of NADPH Oxidase (PHOX), a required step in NET formation. Superoxide has previously been shown to signal for neutrophil activation and increased proinflammatory cytokine production through TLR4, but it is unknown whether it also induces NETs. We hypothesize that in settings of non-infectious inflammation, such as oxidative stress, reactive oxygen species, specifically superoxide, induce NET formation through TLR4.

Methods: WT and TLR4KO neutrophils were treated with xanthine oxidase and its substrate hypoxanthine to generate extracellular superoxide. We inhibited xanthine oxidase by allopurinol and inhibited PHOX by diphenylene iodonium (DPI). We also performed neutrophil depletion and adoptive transfer of TLR4KO neutrophils followed by liver I/R.

Results: In vitro, WT neutrophils exposed to superoxide demonstrated elevated levels of citrullinated Histone H3, a specific NET marker, by western analysis; however, TLR4KO neutrophils expressed only basal cit-H3 despite superoxide treatment. Superoxide-inhibited neutrophils (allopurinol treatment) or PHOX-inhibited (DPI treatment) also expressed only basal cit-H3 compared to treatment with phorbol-myristate-acetate (PMA; positive control) in both WT and TLR4KO neutrophils. Thus, the activity of both TLR4 and PHOX are necessary to complete citrullination of histone H3 and chromatin decondensation. Additionally, superoxide exposure generated characteristic NET fibers in WT neutrophils, but not TLR4KO, as visualized qualitatively by immunofluorescence microscopy and measured quantitatively by mean cytoplasmic spot intensity of extracellular DNA. In vivo, MPO-DNA ELISA, a specific marker for NETs, revealed decreased NET formation in mice adoptively transferred with TLR4KO neutrophils, correlating with protection from liver injury by ALT assay.

Conclusion: In summary, our study demonstrates that superoxide induces NETs, and that WT TLR4 and functional PHOX are required for this process. During liver I/R, ROS can stimulate NETs through TLR4, while its absence precludes NET formation correlating with reduced liver inflammation and injury. Furthermore, our finding that extracellular superoxide induces NETs expands upon previous findings that superoxide activates neutrophils and increases proinflammatory cytokine production. This suggests that products of oxidative stress and damage-associated signals released in liver I/R act via ligand-receptor interactions to promote NET formation.

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