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.

43.07 DPR Decreases TLR4 and MYD88 Expression in the Lung after Resuscitated Hemorrhagic Shock

Posted on December 22, 2014

M. A. Eid2, P. J. Matheson1,2,3, C. D. Downard1,2, R. N. Garrison1,2,3, J. W. Smith2,3  1Robley Rex Veterans Affairs Medical Center,Louisville, KY, USA 2University Of Louisville,Surgery,Louisville, KY, USA 3University Of Louisville,Physiology & Biophysics,Louisville, KY, USA

Introduction: Acute Lung Injury (ALI) leading to Adult Respiratory Distress Syndrome (ARDS) is an early sign of multiple organ dysfunction (MOD) after hemorrhagic shock (HS).  Gut-derived pro-inflammatory factors via mesenteric lymph initiate systemic inflammatory response (SIRS), possibly via lung toll-like receptor 4 (TLR4). Adjunct Direct Peritoneal Resuscitation (DPR) following HS mitigates the inflammatory response.  Deficiencies in downstream pathways from TLR4 (i.e., MYD88 and TRIF) attenuate SIRS by an unknown mechanism.  We hypothesized that DPR would improve lung function in resuscitated HS (HS/CR) by altering levels of TLR4 and 2nd messengers MYD88/TRIF.

 

Methods:   Anesthetized Sprague-Dawley rats were randomly assigned to groups (n=8/group):  1) HS/CR (HS=40% MAP for 60min, CR=shed blood + volumes NS); 2) HS/CR+DPR at time of CR;  3) HS/CR+DPR(120) at 120min post-resuscitation (postRES); 4) Sham (no HS, no CR, no DPR); 5) Sham + DPR at CR; and 6) Sham+DPR(120).  All groups were followed for 4hr postRES.  ELISA was used to measure lung TLR4, MYD88, and TRIF as well as mesenteric lymph and serum LPS.

Results:  HS/CR increased LPS and TLR4 and MYD88 expression in the lung compared to Shams but did not alter TRIF (see Figure).  HS/CR+DPR decreased TLR4 and MYD88 levels but did not alter TRIF levels.  Delayed addition of DPR (HS/CR +DPR(120)) had a similar effect.

Conclusions:  Gut-derived mediators of systemic inflammation can be modulated by peritoneal resuscitation with hypertonic peritoneal dialysis solution to prevent activation of lung inflammatory processes.  These benefits occur when DPR is given immediately at the time of conventional resuscitation or when DPR is delayed by 2 hours after the start of conventional resuscitation.

43.08 MyD88 Inhibition in Limb Ischemia Increases Myofiber Size and Perfusion in TRIFKO Mice

Posted on December 22, 2014

J. Xu1, X. Cui1, E. Tzeng1, U. Sachdev1  1University Of Pittsburgh,Vascular Surgery,Pittsburgh, PA, USA

Introduction: We have shown that mice with genetically absent MyD88, a critical mediator of TLR4 signaling, showed less ischemic damage and inflammation than controls after femoral artery ligation.  Surprisingly, mice lacking TRIF, another important mediator of TLR4 signaling demonstrated necrosis rather than protection at the same time point. These earlier findings suggested that MyD88 and TRIF played complimentary roles in the recovery from limb ischemia. In order to further test this theory, we treated control C57B6 mice as well as TRIFKO with a peptide inhibitor of MyD88 to test the hypothesis that unopposed MyD88 inflammation in TRIFKO mice hindered muscle recovery after femoral ligation.

Method: C57B6 mice and TRIFKO mice were injected with 25 micrograms of peptide inhibitors to MyD88 or a nonspecific target one hour before the initiation of femoral artery ligation on the right hindlimb. On the left, the femoral vessels were exposed but not ligated. Laser Doppler Perfusion Imaging (LDPI) was performed after one week to assess perfusion recovery, expressed as a ratio of right to left flow. H and E was performed on ischemic muscle samples and the average area of each regenerating myocyte, identified by centrally located nuclei, was compared among groups.

Results:  TRIFKO mice treated with a MyD88 inhibitor demonstrated an improvement in perfusion by LDPI 7 days after ligation (Figure 1). Neither control C57B6 mice nor mice treated with control peptide inhibitor demonstrated similar improvements in perfusion to the ischemic limb at the same time point.  Similarly, the mean size of regenerating myocytes was significantly higher in TRIFKO mice treated with MyD88 inhibitor but not control inhibitor (295.12 ± 14.98 vs. 235.00 ± 14.72 micrometers; MyD88 inhibitor vs. control inhibitor, p=0.038, N=3-4/group). Similar improvements were not demonstrated in C57B6 mice treated with the MyD88 inhibitor.

Conclusion: Our data suggest that in the absence of TRIF, MyD88 activity may compromise muscle regeneration after ischemia.  Blocking its activity with a peptide inhibitor improves perfusion recovery and increases regenerating myofiber size after one week.  These results further support the hypothesis that TRIF attenuates MyD88 activity, acting to balance the deleterious inflammatory effects of intact MyD88 signaling in limb ischemia.  

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.

43.10 Adeno-Thioredoxin- 1 Delivery Improves Neovascularization in a Murine Model of Hind Limb Ischemia.

Posted on December 22, 2014

M. T. Rishi1,2, I. A. Shaikh1,2, V. Selvaraju1, M. Thirunavukkarasu1, J. Palesty2, N. Maulik1  1University Of Connecticut Health Center,Molecular Cardiology And Angiogenesis Laboratory, Department Of Surgery,Farmington, CT, USA 2Saint Mary’s Hospital,Stanley J. Dudrick Department Of Surgery,Waterbury, CT, USA

Introduction: There is a growing interest in developing novel therapeutic modalities at a molecular level for the treatment of peripheral arterial disease (PAD). We have previously shown that Thioredoxin -1 (Trx-1), which is a cytosolic 12-kDa redox protein, enhances neovascularization and reduces ventricular remodeling during chronic myocardial infarction. Here we aim to investigate the role of this protein in a murine hind limb ischemia (HLI) model.

Methods: Adult 8-12 week old C57Bl/6 mice were divided into two groups: (1) adeno-thiroedoxin-1 gene treatment group (Ad-Trx-1) and (2) control group (Ad-LacZ). The mice in Ad-Trx-1 group underwent right femoral artery ligation to create a murine model of HLI. Immediately after surgery, adeno-thioredoxin -1 in a concentration of 1×10⁹ PFU was injected in both semimembranosus and gastrocnemius muscles of the right leg whereas the left leg was used as an internal control. The mice in Ad-LacZ group also underwent right femoral artery ligation but instead received similar concentration of Ad-LacZ at the same muscle sites. The two groups underwent serial laser doppler imaging (LDI) pre-operatively and post-operatively for 28 days to assess hind limb perfusion. Immunohistochemistry and ELISA were performed on post-operative day 4 to determine the expression for various key angiogenic proteins. 

Results: Mice in the Ad-Trx-1 group showed a significantly increased perfusion ratio on postoperative day 21  [0.893±0.067 (n=10) vs. 0.593±0.065 (n=10); p<0.05] and day 28 [0.908±0.081 (n=08) vs. 0.660±0.057 (n=10); p<0.05] [Figure 1] and a higher motor function score on post-operative day 7 [3.1±0.233 (n=10) vs. 2.1±0.276 (n=10); p<0.05], day 14 [3.9±0.233 (n=10) vs. 2.8±0.249 (n=10); p<0.05], day 21 [4.9±0.1 (n=10) vs. 3.7±0.213 (n=10); p<0.05] and day 28 [4.875±0.125 (n=8) vs. 3.8±0.249 (n=10); p<0.05] as compared to mice in Ad-LacZ group. Four days after femoral artery ligation, Ad-Trx-1 group showed increased Vascular Endothelial Growth Factor (VEGF) expression by immunohistochemical analysis and ELISA [38.48±7.165 (n=4) vs. 21.70±2.560 (n=5); p<0.05] as compared to Ad-LacZ group. Ad-Trx-1 group also showed increase expression of Flt-1 and Angiopoietin -1 proteins as compared to Ad-LacZ group by immunohistochemical analysis. 

Conclusion: Taken together, our study demonstrates that the adeno-thioredoxin-1 gene delivery enhances blood perfusion and increases angiogenic protein expression in a murine hind limb ischemia model. We hope that this molecule can be a future potential target for clinical trials and subsequently drug therapy in peripheral vascular disease management.

 

 

44.01 Cardiac Progenitor Cell Recruitment Inhibition During Fetal Response to MI Leads to Scar Formation

Posted on December 22, 2014

C. Zgheib1,2, J. Xu1,2, M. Allukian3, M. W. Morris2, R. C. Caskey3, J. Hu1,2, J. H. Gorman3, R. C. Gorman3, K. W. Liechty1,2  1University Of Colorado Denver,School Of Medicine – Department Of Surgery,Aurora, CO, USA 2Colorado Children’s Hospital,Department Of Surgery,Aurora, CO, USA 3University Of Pennsylvania,The Perelman School Of Medicine,Philadelphia, PA, USA

Introduction: The normal fetal response to myocardial infarction (MI) results in regeneration of functional myocardium.  In contrast, the adult response results in infarct scar formation and progressive decline in cardiac function due to scar remodeling.  We have shown that inhibition of stromal-derived factor-1α (SDF-1α) in the fetal infarct blocks the recruitment of cardiac progenitor cells and results in a lack of regeneration, similar to the adult response.  We hypothesized that this lack of regeneration is due to fetal infarct scar formation, increased collagen gene expression, and increased collagen remodeling.

Methods: To test this hypothesis, 20% apical infarcts were created in adult and fetal sheep by LAD ligation.  In a subset of fetal sheep, the infarcts were injected with a lentivirus containing the SDF-1α inhibitor transgene.  Animals were harvested 3 or 30 days following infarction.  Echocardiography was performed to assess cardiac function and infarct size.  Histology was used to assess scar formation.  Real-time PCR was used to assess collagen (Col 1α1) and matrix metalloproteinase-9 (MMP-9) gene expression.

Results: At 3 days following MI, both fetal and adult hearts demonstrated significant infarcts. At 30 days, the adult heart demonstrated infarct expansion and decreased function.  In contrast, the fetal hearts demonstrated resolution of the akinetic myocardium and restoration of cardiac function.  However, SDF-1α inhibition transformed the regenerative fetal response into a reparative response similar to the adult.  At 30 days, fetal hearts treated with the SDF-1α inhibitor demonstrated a significant increase in infarct size as well as scar formation by histology.  In addition, SDF-1α inhibition resulted increased collagen and MMP-9 gene expression.

Conclusion: The fetal regenerative response to myocardial infarction is dependent on the recruitment of cardiac progenitor cells mediated by SDF-1α.  These recruited progenitor cells may create an environment conducive to restoration of functional myocardium by altering collagen gene expression and remodeling.  These novel findings provide the first evidence that SDF-1α plays a role in the regulation of fibrosis or scar formation.  Further studies are need to define the role of progenitor cell recruitment in promoting fetal cardiac regeneration.

 

44.02 Attenuation of Abdominal Aortic Aneurysms with Mesenchymal Stem Cells from Different Genders

Posted on December 22, 2014

J. P. Davis1, M. Salmon1, N. Pope1, G. Lu1, A. Meyer1, G. Su1, G. Ailawadi1, G. R. Upchurch1  1University Of Virginia,Surgery,Charlottesville, VA, USA

Introduction:

As no medical therapies are yet available to slow abdominal aortic aneurysm (AAA) growth, this study sought to investigate the effect of different genders of bone marrow derived mesenchymal stem cells (MSCs) on AAA growth in a murine AAA model. Given the decreased rate of AAA in women, it is hypothesized that female MSCs would attenuate AAA growth more so than male MSCs.

 

Methods:

Aortas of 8- to 10-week-old male C57Bl/6 mice were perfused with purified porcine pancreatic elastase (0.35 units/ml) to induce AAA formation. MSCs were harvested from bone marrow of femurs of 7- to 8-week-old male and female C57Bl/6 mice and male and female red fluorescent protein homozygous mice (RFP+/+) and isolated using standard protocols. MSCs from male and female mice were dosed via tail vein injection (3 million cells per dose, 500 µL of volume per injection) on post-aortic perfusion days 1, 3, and 5. Aortas were harvested after 14 days. Video micrometry was used to measure AAA phenotypic characteristics. Fluorescent staining was performed on aortic tissue and evaluated with confocal microscopy. Cytokine arrays were performed on aortic tissue. Groups were compared using an ANOVA and a Tukey’s post hoc test.

 

Results:

Mean aortic dilation in the elastase group was 121±13.8% (mean±SD), while male MSC inhibited AAA growth (*87.8±22.8%, P=0.0014) compared to elastase. However, female MSCs showed the most marked attenuation of AAA growth when compared to elastase (**75.2±25.9% P= 0.0008, Figure). Convergence of confocal microscopy channels revealed RFP+/+ MSCs of both genders integrated within the aortic wall. Tissues treated with either male or female MSCs revealed significantly decreased levels of Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1)(males p=0.0011, females p=0.0003 compared to elastase) and Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) (males p=0.003, females p<0.0001 compared to elastase). Pro-inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), and macrophage chemotactic protein-1 (MCP-1) were only decreased in tissues treated with female MSCs (p=0.001, p=0.01, and p<0.001, respectively when compared to elastase).

 

Conclusions:

These data document that MSCs attenuate AAA growth in the murine model, and MSCs integrate into the aortic wall after treatment. Furthermore, female MSCs and male MSCs inhibit proinflammatory cytokines at varying levels.  The effects of MSCs on aortic tissue offer a promising insight into biologic therapies for future medical treatment of AAAs in humans.

44.03 Chronic Stress-Induced Impaired Lung Healing Following T/HS is Reversed by Mesenchymal Stem Cells

Posted on December 22, 2014

A. V. Gore1, L. E. Bible1, K. J. Song1, A. M. Mohr1, D. H. Livingston1, Z. C. Sifri1  1Rutgers New Jersey Medical School,Surgery,Newark, NJ, USA

Introduction:  Rat lungs undergo full histologic recovery one week following unilateral lung contusion (LC).  This healing is impaired when LC is followed by hemorrhagic shock (HS), and even more so in the setting of chronic stress (CS).  We hypothesize that injecting mesenchymal stem cells (MSC) could reverse LCHS/CS impaired lung healing.

Methods:  Male Sprague-Dawley (SD) rats (n=5-6/group) underwent LCHS/CS with or without the addition of a single iv dose of 5 x 106 SD rat MSCs at the time of resuscitation.  Rats experienced two hours of restraint stress on days 1-6 following injury and were sacrificed on day 7.  Lung histology was scored according to a well-established lung injury score (LIS) to grade injury.  Components of the LIS include interstitial and pulmonary edema, alveolar integrity, and inflammatory cells/high power field (hpf) averaged over 30 fields.  Total scores range from 0-11.  Data expressed as mean ± SD; analyzed by ANOVA followed by Tukey’s multiple comparison test. 

Results:  Seven days following insolated LC, LIS has returned to 0.8 ± 0.4, while it remains elevated with the addition of HS (3.7 ± 0.8) and further elevated with combined LCHS/CS (7.2 ± 2.2), as previously reported.  Addition of MSC to LCHS/CS decreased LIS to 2.0 ± 1.3 and decreased all subgroup scores (inflammatory cells, interstitial and pulmonary edema, and alveolar integrity) significantly as compared to LCHS/CS.  

Conclusion:  In this rat lung contusion model, the severe impairment of lung healing seen seven days following LCHS/CS is reversed by MSCs given early at the time of resuscitation.  This improvement in healing is associated with a decrease in the number of inflammatory cells and decreases in lung edema scores. Further study into timing of administration and mechanisms by which MSC improve wound healing is warranted. 

 

44.04 Angiopoietin-1 Improves Endothelial Progenitor Cell Dependent Neovascularization in Diabetic Wounds

Posted on December 22, 2014

S. Balaji1, R. Ranjan1, C. Moles1, N. Hann1, T. M. Crombleholme1,2, S. G. Keswani1  1Cincinnati Children’s Hospital Medical Center,Pediatric General Thoracic And Fetal Surgery,Cincinnati, OH, USA 2Children’s Hospital Of Colorado,Center For Children’s Surgery,Aurora, CO, USA

Introduction: The diabetic wound healing phenotype is in part characterized by impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Over expression of angiogenic growth factors to improve EPC recruitment and wound healing is a novel strategy to treat diabetic wounds. Angiopoetin-1 (Ang1) is a potent mobilizer of EPCs from the bone marrow (BM) and we have shown that Ang1 has direct effects on reepithelialization. A suggested mechanism for EPC mobilization from the BM is mediated through MMP9 and stem cell factor (SCF). Taken together we hypothesize that Ang1 overexpression in diabetic wounds will recruit EPCs and improve neovascularization and wound healing.

Methods: To track BM derived EPCs we used an endothelial lineage BM labelled murine model with diabetes. FVBN mice were lethally irradiated and subsequently reconstituted with BM from syngeneic TIE-2/LacZ donor mice. Diabetes was induced with streptozotocin (STZ). 8 mm wounds were created in BM transplanted mice (BMT) (n=12) and treated with 10 8 PFU of AdAng1, AdGFP or PBS. Wounds were harvested at day 7 post wounding and analyzed for epithelial gap, vessel density and Tie-2/LacZ positive EPCs recruitment. Serum levels of VEGF, proMMP9 and SCF were assessed by ELISA. To determine if Ang1 effects were EPC dependent a simiar experiment was conducted in EPC mobilization deficient mice (MMP9 KO). Data presented as mean+/-SEM, p-values by ANOVA. 

Results: All mice survived at 4 weeks post BMT. Mice with STZ-induced diabetes had blood glucose >350mg/dl.  Ang1 resulted in significantly improved reepithelialization (Ang1 2.3+/-.2mm; GFP 3.9+/-.2; PBS 4.0+/-.1 p<.0001), neovascularization (Ang1 6.8+/-.3Caps/Hpf; GFP 3.0+/-.4; PBS 2.9+/-.3 p<.0001) and EPC recruitment (Ang1 5.3+/-.4 EPCs/Hpf; GFP 2.1+/-.3; PBS 2.2+/-.3 p<.0001) in diabetic BMT wounds at day 7. Ang1 treatment resulted in increased levels of proMMP9 (Ang1 9.7+/-.8 ng/ml; GFP 6.3+/-.9; PBS 6.4+/-.4 p<.01) and SCF (Ang1 265+/-28 pg/ml; GFP 119+/-16; PBS159+/-12 p<.001) but had no effect on VEGF. Peripheral blood EPC (CD34+/Cd133+/Flk1+) counts at day 3 post wounding using FACS demonstrated impaired EPC mobilization in MMP9 KO mice compared to wild type controls (MMP9 KO 0.66+/-0.02; WT 1.1+/-0.05 p<0.001), which was rescued by SCF administration (MMP9 KO+SCF 1.23+/-0.09; WT 1.1+/-0.05 p=ns), validating this model for EPC loss-of-function mechanistic studies. In MMP9 KO mice, AdAng1 similarly accelerated reepithelialization (Ang1 3.2+/-.1mm; GFP 4.1+/-.2; PBS 3.8+/-.2; p<.05), but had no effect on neovascularization (Ang1 4.1+/-.5 Caps/HPF; GFP 3.9+/-.4; PBS 3.9+/-.6).

Conclusion: Our results demonstrate that Ang1 results in improved neovascularization which is dependent on EPC recruitment and that it has direct effects on wound reepithelialization. These data may represent a novel strategy to correct the impaired diabetic neovascularization phenotype and improve diabetic wound healing. 

 

44.05 HMGB1 Released by Platelets Leads to Microvascular Thrombosis Following Trauma and Hemorrhage

Posted on December 22, 2014

M. D. Neal1, J. Markel1, B. S. Zuckerbraun1, J. L. Sperry1, P. Loughran1, T. R. Billiar1  1University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA

Introduction:  Platelet dysfunction has been identified as a major contributor to the pathogenesis of trauma and hemorrhage.  Recently, high-mobility group protein 1 (HMGB1), an endogenous danger signal and mediator of sterile inflammation has been shown to be released from platelets.  However, the response of platelets to HMGB1 and the specific function of platelet derived HMGB1 in sterile injury remain unexplored.   

Methods:  HMGB1 in human and murine platelets was identified using ELISA, flow cytometry, and immunostaining.  Mice were treated with recombinant HMGB1 (rHMGB1) to study the exogenous effect of HMGB1 on platelets and platelet function was measured by aggregometry and thromboelastography (TEG).  Ferric chloride induced thrombosis was used to study the specific role of HMGB1 in thrombosis.  Using cre-loxp technology, we generated mice specifically lacking HMGB1 on platelets (HMGB1Pf4-cre).  HMGB1Pf4-cre and wild-type (WT) mice were subjected to a model of trauma and hemorrhagic shock consisting of laparotomy, liver crush injury, and hemorrhage followed by resuscitation (THS-R) with tissue harvest for analysis of injury.  Aggregometry was performed on isolated platelets after trauma.  

Results:  Administration of rHMGB1 to mice led to increased platelet aggregation, microvascular thrombosis, and hypercoagulability as measured by TEG.  THS-R resulted in similar platelet hyper-aggregation and sequestration of platelets into lung and liver in WT mice.  Platelets released HMGB1 following activation and sterile injury.  Strikingly, selective deletion of HMGB1 from platelets (HMGB1Pf4-cre) resulted in platelets that were resistant to hyperaggregation and sequestration with reduced adherence to microvascular endothelium.  HMGB1Pf4-cre mice had markedly reduced liver injury compared to WT as measured by serum AST (850 ± 33.2 IU/L vs 505 ± 61.7 IU/L, p=0.02).   Additionally, HMGB1Pf4-cre mice were protected from lung and liver injury as measured by histologic scoring, with markedly reduced microvascular thrombosis vs WT (22.5 ±2.2 vs 5.1 ±1.4, p<0.01).  Analysis of thrombi from WT and HMGB1Pf4-cre mice revealed that platelets are the major source of HMGB1 in clot.

Conclusion:  Platelet derived HMGB1 is an essential mediator of platelet aggregation following trauma and hemorrhage.  Deletion of HMGB1 from platelets nearly eliminated microvascular thrombosis and decreased organ injury following THS-R.  Modulating HMGB1 release from platelets may represent a novel therapeutic target for preventing organ injury after trauma.

 

44.06 Amitriptyline Reduces Endothelial Damage from Packed Red Blood Cell-Derived Microparticles

Posted on December 22, 2014

P. L. Jernigan1, R. S. Hoehn1, J. Sutton1, E. Midura1, T. Johannigman1, C. C. Caldwell1, M. J. Edwards1, E. Gulbins1,2, T. A. Pritts1  1University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA 2University Of Duisburg-Essen,Department Of Molecular Biology,Essen, NORTH RHINE-WESTPHALIA, Germany

Introduction:  Transfusion with older packed red blood cell (pRBC) units during trauma resuscitation is associated with lung injury and increased mortality. Our recent work implicates pRBC-derived microparticles (MPs) in this process. Ceramide, a cell membrane-based sphingolipid, can be formed by the enzyme acid sphingomyelinase (Asm) and act as a signaling agent to alter cell membranes. The potential role of the Asm/ceramide system in mediating microparticle formation and lung injury from stored pRBC units is unknown. We hypothesized that inhibiting Asm in stored pRBC units could decrease the harmful effects of pRBC-derived MPs. 

Methods:  Murine pRBC units were prepared from healthy donors, treated with the Asm inhibitor Amitriptyline (AT; up to 250 µM) or PBS (vehicle) and stored under standard blood banking conditions for up to 14 days. MPs were isolated and counted with Nanoparticle Tracking Analysis immediately prior to use. Healthy mice underwent hemorrhage and resuscitation that included equal concentrations of MPs isolated from AT- or vehicle-treated pRBC units and lungs were collected for analysis. To study specific effects of MPs on endothelial cells, murine hemangioendothelioma (EOMA) cells were incubated for thirty minutes with equal numbers of MPs from AT- or vehicle-treated pRBCs. Cells were then fixed and stained for the tight junction proteins ZO-1 and occludin. 

Results: Treatment with amitriptyline reduced Asm activity and ceramide levels in MPs isolated from the pRBC units. Mice that underwent hemorrhage followed by resuscitation with MPs isolated from vehicle-treated pRBC units demonstrated increased inflammatory cell recruitment to the lung, alveolar wall thickening, and decreased pulmonary ZO-1 and ZO-2 expression relative to baseline, consistent with acute lung injury. These changes were significantly attenuated in mice resuscitated with equal numbers of MPs from AT-treated pRBC units. EOMA cells treated with MPs from aged vehicle-treated pRBCs showed a significant reduction in the tight junction proteins ZO-1 (Figure A) and occludin relative to untreated cells or those treated with MPs from fresh (unstored) pRBCs, consistent with tight junction disruption. This decrease in ZO-1 and occludin was abrogated in cells treated with MPs isolated from AT-treated pRBCs (Figure B and C).

Conclusion: Microparticles shed by stored pRBCs mediate lung injury after resuscitation, with disruption of endothelial cell tight junctions. Treating stored pRBCs with the Asm inhibitor Amitriptyline mitigates this effect. These data suggest that Asm inhibition may ameliorate harmful aspects of pRBC storage and reduce lung injury after transfusion.

44.07 Loss of MuRF-3 Up-regulates Cardiac PPAR-β Activity and Exaggerates Diabetic Cardiomyopathy

Posted on December 22, 2014

M. T. Quintana1, J. He3, M. S. Willis3, W. E. Stansfield2  1University Of North Carolina At Chapel Hill,Department Of Surgery,Chapel Hill, NC, USA 2University of Toronto,Department Of Surgery, Division Of Cardiovascular Surgery,Toronto, Ontario, Canada 3University Of North Carolina At Chapel Hill,Department Of Pathology & Laboratory Medicine,Chapel Hill, NC, USA

Introduction:
Diabetic cardiomyopathy is characterized by structural and functional alterations leading to cardiac hypertrophy and heart failure. Alterations include metabolic disturbances in energy metabolism, increased lipid oxidation, intramyocardial triglyceride accumulation, and reduced glucose oxidation. These metabolic changes are regulated in part by peroxisome proliferator activating receptor (PPAR) signaling and subsequent changes in fatty acid metabolism. We recently identified that mice lacking ubiquitin ligase Muscle RING Finger 3 (MuRF3-/-) have increased cardiac PPAR-β activity in vivo. We hypothesized that challenging MuRF3-/- mice with a high-fat diet, where lipids act as PPAR ligands, would enhance the severity of diabetic cardiomyopathy. 

Methods:
MuRF3-/- mice and strain-matched controls were fed a standard chow or high-fat diet for 26 weeks. Body weight, serum insulin, serum glucose, and fasting cholesterol and triglyceride levels were measured every two weeks and echocardiography was performed every three weeks. CPT1b, a key regulator of fatty acid metabolism downstream of PPAR-β in the diabetic cardiomyopathy pathway, was evaluated with real-time PCR.

Results:
MuRF3-/- mice have less circulating glucose after 26 weeks on high-fat diet (HFD) as compared to strain matched controls (168.8±6.2 vs. 240.2±15.5 mg/dL, P<0.05) and increased fasting circulating insulin levels relative to controls (4.7±0.3 vs. 3.4±0.2 ng/ml, P<0.05). Knockout mice do not show differences in fasting total cholesterol compared to wild-type mice at baseline (167±7.0 vs. 141±15.5 mg/dL, P=0.21) or 26 weeks HFD (162±24.3 vs. 151±8.0 mg/dL, P=0.65). There are no differences in serum triglyceride levels between knockout or wild-type mice at baseline (118±11.0 vs. 101±4.3 mg/dL, P=0.16) or after 26 weeks HFD (126±13.7 vs. 131±5.2 mg/dL, P=0.72). By echocardiography, MuRF3-/- mice have profoundly depressed fractional shortening (FS) at 6 weeks HFD compared to wild-type mice (43.6±6.1% vs. 49.2±3.5%, P<0.05). Wild-type mice do not display systolic dysfunction until 26 weeks HFD (FS 43.6±1.8 %). CPT1b mRNA expression is increased in MuRF3-/- mice (ΔΔct 3.57±1.1 in MuRF3-/- vs. 1.00±0.2 in controls, P<0.05). 

Conclusion:
Mice lacking MuRF3 demonstrate exaggerated cardiomyopathy in the presence of high-fat diet. Increased PPAR-β activity, as well as increased CPT1b, are known contributors to diabetic cardiomyopathy. Taken together, these findings help corroborate a novel role for MuRF3 in protecting against the development of diabetic cardiomyopathy.
 

44.08 Role of Sphingosine-1-phosphate in Inflammation-induced Lymphangiogenesis

Posted on December 22, 2014

L. J. Fernandez1, W. Huang1, K. P. Terracina1, M. Nagahashi3, A. Yamada2, T. Aoyagi4, S. Spiegel5, K. Takabe1,5  1Virginia Commonwealth University,Surgical Oncology,Richmond, VA, USA 2Yokohama City University,Surgery,Yokohama, , Japan 3Nigata University,Digestive And General Surgery,Nigata, , Japan 4Chiba University,Surgery,Chiba, , Japan 5Virginia Commonwealth University,Biochemistry And Molecular Biology,Richmond, VA, USA 6Chang Gung Memorial Hospital,Taipei, , Taiwan

Introduction:
There is growing evidence that sphingosine-1-phosphate (S1P), a pleiotropic bioactive sphingolipid metabolite formed inside cells by two closely related sphingosine kinases, SphK1 and SphK2, is involved in inflammation and cancer. Previously, we have reported the involvement of S1P and SphK1 in colitis and colitis-associated cancer (CAC) utilizing SphK2 knockout mice in Cancer Cell. S1P has been found to play an important role in inflammation and CAC through persistent activation of the NF-kappaB/IL-6/STAT3 axis by S1P. In this current study we sought to investigate the role of S1P in lymphangiogenesis and lymphatic vessel dilatation in a DSS colitis model.

Methods:
SphK2 knockout mice were kindly provided by Dr. Proia, NIH. Dextran Sulfate Sodium (DSS) in drinking water was used to generate accolitis model. Lymphangiogenesis and lymphatic vessel dilatation were determined by micro-vessel density and the area of the vessel with both IHC and Immunofluorescence on colon and mesenteric lymph node (MLN) samples. As methods of blocking S1P signaling, we used SK1-I, a SphK1 specific inhibitor, W146, a S1PR1 specific inhibitor, and FTY720, a S1PR1 functional antagonist

Results:
When comparing wild type (WT) and SphK2 knock out (KO) mice, SphK2 KO mice were shown to overexpress SpkK1 resulting in higher extracellular levels of S1P. We have observed that SphK2 KO mice demonstrated stronger mucosal damage compared to WT mice by histology colitis severity score when treated with DSS. Both lymphangiogenesis and lymphatic vessel dilatation were increased in SphK2 KO after DSS treatment in both colon and MLN. Morphologically, SphK2 KO mice demonstrated increased lymphatic vessel vasculature when compared to WT but the difference was much higher with DSS treated mice, which was evident both in the colon and in the MLNs. The inhibition of SphK1 by SK1-I or S1PR1 by W146 or FTY720 resulted in a significant decrease of lymphangiogenesis and dilatation of lymphatic vessels in DSS induced colitis

Conclusion:
We found that SphK1 overexpressing, thus S1P producing, animals demonstrated severe colitis with S1P mediated enhancement of lymphangiogenesis and lymphatic vessel dilatation. Considering the fact that S1P signaling blockade significantly suppressed lymphangiogenesis and lymphatic vessel dilatation, we conclude that S1P plays a critical role in inflammation-induced lymphangiogenesis and lymphatic vessel dilatation
 

44.09 DPR Up-regulates VEGF-A and Reduces Edema And Acute Lung Injury in Resuscitated Hemorrhagic Shock

Posted on December 22, 2014

M. A. Wilson2, S. A. Matheson2, P. J. Matheson1,2,3, C. D. Downard1,2, R. N. Garrison1,2,3, J. W. Smith1,2,3  1Robley Rex Veterans Affairs Medical Center,Louisville, KY, USA 2University Of Louisville,Surgery,Louisville, KY, USA 3University Of Louisville,Physiology & Biophysics,Louisville, KY, USA

Introduction: Acute Lung injury (ALI) following hemorrhagic shock (HS) is a life threatening complication. VEGF-A levels correlate with increased pulmonary capillary leak and deranged vascular control mechanisms in ALI. Direct Peritoneal Resuscitation (DPR) increases microvascular perfusion and reduces end organ ischemia in resuscitated hemorrhagic shock (HS/CR).  DPR’s effects on pulmonary injury in HS/CR have not been studied.  Our goal was to determine DPR effects on the lung injury and VEGF expression following HS/CR.

Methods: Anesthetized Sprague-Dawley rats were randomly assigned to groups (n=8/group):  1) Sham (no HS, no CR, no DPR); 2) HS/CR (HS=40% MAP for 60min, CR=shed blood + volumes NS); 3) HS/CR+DPR at time of CR.  All groups were followed for 4hr post RES.  Multi-array Luminex was used to measure lung cytokine levels and wet-dry weights were used to evaluate edema formation during resuscitation. PCR SuperArrays were used to assess cellular mRNA levels.  Histologic and immunohistochemistry was used to assess pulmonary injury. Pulmonary artery perfusion was evaluated by colorimetric microsphere technique.

Results: DPR enhanced pulmonary blood flow, reduced pulmonary edema, and reduced histologic evidence of pulmonary injury (see Table). Adjunct DPR increased lung perfusion by 111% compared to HS/CR alone (*P<0.01).  HS/CR caused lung edema, which was restored to Sham levels in HS/CR+DPR.  HS/CR decreased VEGF-A protein levels and adjunct DPR partially restored VEGF-A expression.  VEGF-A mRNA levels in HS/CR were down-regulated compared to Sham and adjunct DPR up-regulated VEGF-A mRNA compared to Sham.

Conclusions: Pulmonary edema formation stimulates VEGF-A expression in other forms of ALI, but we observed elevated VEGF expression during reduced pulmonary edema and injury following adjunct DPR treatment.  Also, VEGF is thought to preserve endothelial cell function and control, which was consistent with increased pulmonary artery perfusion.  ALI-associated edema after resuscitated HS might not play a key role in lung edema in this model.  Our data (i.e., VEGF-A protein and mRNA expression) suggest that normalized levels of VEGF are protective of lung perfusion following resuscitated hemorrhagic shock in this model.

40.01 Are Surgical Residency and Parenthood Compatible?

Posted on December 22, 2014

C. Kin1, M. Esquivel1, C. Mueller1  1Stanford University,Surgery,Palo Alto, CA, USA

Introduction:  Surgical residents commit to a minimum of five years of intensive training during their prime reproductive years. Our hypothesis is that while all surgical residents face significant barriers that prevent them from starting families, there are gender differences in their attitudes towards having children. The aim of this study is to identify the concerns and challenges of surgical residents with regards to having children. 

Methods:  We administered a 32-question anonymous online survey to all residents in general surgery and surgical subspecialties at a tertiary care academic medical institution. Participants were compensated with a $10 gift certificate. Chi-square and Fisher’s exact tests were used to determine if there was a significant difference between groups, and p<0.05 was considered significant.

Results: Of 171 surveys sent, 83 were started and 80 completed; 53% of respondents were men and 46% were women. Men and women were similar in age distribution (54% aged 31-35, 41% aged 26-30), as well as proportions in long-term relationships (75% vs 79%) and proportions with children (18% vs 21%), with most parents having one child. Male and female residents are similar in their concerns that they had inadequate time, money, and childcare resources to have children.(Table 1) Women are more likely to worry that having children would negatively affect the way they were perceived professionally (55% vs 16%, p=0.0002), make them a burden on their colleagues (84% vs 39%, p<0.0001), and negatively impact their future careers (53% vs 23%, p=0.005). Women are also more likely to believe that there is no optimal professional time to have a child (74% vs 43%, p=0.005). The majority of childless residents agreed that they are deferring having children because of their jobs, and 73% feel anxious when they think about having children. Childless female residents are more likely to be worried about the possibility of not ever having children (77% vs 44%, p=0.008). Only a third of residents have discussed the topic of having children with a mentor, and of those who have not discussed it, women are more likely than men to want to have that discussion with a mentor (56% vs 26%, p=0.04). 

Conclusion: The scarce financial resources and time commitment inherent to surgical training are barriers that prevent many residents from starting families and pose great difficulties for residents who do have children. Female surgical trainees are particularly worried about work-family conflicts. Residency programs and mentors should be aware of these stressors and creative solutions are needed to promote the welfare of their surgical trainees. 

 

40.02 Practice Administration Training Needs of Recent General Surgery Graduates

Posted on December 22, 2014

M. Klingensmith1, T. H. Cogbill3, K. Samonte2, A. Jones2, M. Malangoni2  1Washington University,Surgery,St. Louis, MO, USA 2American Board Of Surgery Inc,Philadelphia, PA, USA 3Gundersen Health System,Surgery,LaCrosse, WI, USA

Introduction:  Practice administration education and experience during surgery residency is limited and highly variable among residency programs. To gain understanding into the current status of practice administration training, a survey of recent General Surgery residency (GS) graduates was undertaken. This was compared to results from a survey of GS program directors (PD).

Methods:  All US allopathic GS   graduates who completed residency  from 2009- 2013 (n= 5194) were anonymously surveyed by the American Board of Surgery (ABS) to assess opinions  regarding their desire to have received more instruction and experience  during residency in the following practice administration areas: coding, contract negotiations, practice management, insurance billing, billing the uninsured, liability, insurance for one’s own practice,  and retirement planning. Surveys were distributed by mail in November 2013 with up to two follow up mailings to non-respondents.  General Surgeons were defined as those who did not pursue fellowship training; specialist surgeons (SS) completed additional training following GS residency. Separately, all GS residency PDs were surveyed regarding the inclusion of practice administration education in their residency programs.

Results: There were 3354 respondents to the GS graduate survey (response rate 68%). GS comprised 876 of the total respondents (26%) with SS accounting for the remaining 74%.     The vast majority of all respondents desired more training in all areas of practice administration that were queried: coding instruction (desired by 86%), contract negotiations (84%), practice management (83%), insurance billing (82%), billing those uninsured (77%), liability (76%), insurance for one’s own practice (75%), and retirement planning (72%). There were no significant differences in the degree to which these areas of instruction were desired among graduate year cohorts, residency program type or current practice setting (academic vs community). However, GS tended to have greater desire for this training than SS in many but not all content areas queried. The GS PD survey had a response rate of 68% (171 of 252 programs). Among respondents, only 28% of programs included practice administration as part of the residency curriculum.

Conclusion: 

Despite increased accreditation and ABS requirements for GS residency programs, there is no mandate for trainees to receive practice administration education and this topic is seldom included in the curriculum.  However, such skills are highly desired and needed by program graduates in a range of practice types and  locations. This large survey of recent GS residency graduates indicates a clear and desired need for an improved curriculum and experience in practice administration topics during residency. Steps should be taken to address this educational gap.

 

 

40.03 YouTube is the Most Frequently Used Educational Video Source for Surgical Preparation

Posted on December 22, 2014

A. K. Rapp1, M. G. Healy2, M. E. Charlton3, M. E. Rosenbaum4, M. R. Kapadia2  1University Of Iowa,Carver College Of Medicine,Iowa City, IA, USA 2University Of Iowa,Department Of Surgery,Iowa City, IA, USA 3University Of Iowa,College Of Public Health,Iowa City, IA, USA 4University Of Iowa,Department Of Family Medicine,Iowa City, IA, USA

Introduction: In this technology-driven era, medical professionals have instant access to videos of surgical procedures. In addition to reading and peer consultation, a plethora of online videos are available to surgeons preparing for surgical cases. The purpose of this study was to evaluate the surgical preparation methods of medical students, residents, and attending surgeons, with special attention to video usage.

Methods: Anonymous paper surveys were distributed to fourth-year medical students pursuing general surgery, general surgery residents, and attending surgeons in the Department of Surgery at a single academic medical center following IRB approval. Information collected included demographics and surgical preparation methods, focusing on video usage. Participants were questioned regarding frequency and helpfulness of video usage; video sources most utilized; and preferred preparation methods between videos, reading, and peer consultation. Chi-square and Fisher’s exact tests were used to compare learner (medical student and resident) and attending responses.

Results: The overall response rate was 91%, which included 42 leaners and 36 attendings. Residents (n=33) from each clinical year (1-5) were represented, and attending experience ranged from 1-36 years (mean=11 years). 90% of all respondents reported using videos for surgical preparation, with no significant difference between learners (95%) and attendings (83%). The mean video helpfulness rating was 3.5 (range 1-5; 1=not helpful, 5=very helpful). Of respondents who perform laparoscopic procedures (n=60), all used videos, whereas only 55% of those who do not perform laparoscopic procedures used videos (p<0.0001). Regarding surgical preparation methods overall, most learners and attendings reported reading (63% versus 78%, p=NS) and some reported watching videos (64% versus 44%, p=NS); however, attendings more often utilized peer consultation compared to learners (50% versus 24%, p<0.02).

Among the 90% of respondents that reported using videos, the most commonly utilized video source was YouTube (86%). Learners and attendings used different video sources (see Figure): learners used YouTube and the Surgical Council on Resident Education (SCORE) Portal more frequently than attendings (YouTube: 95% versus 73%, p<0.05; SCORE: 25% versus 7%, p<0.05); however, attendings were more likely than learners to use society webpages and commercial videos (society: 67% versus 38%, p<0.03; commercial: 27% versus 5%, p<0.02).

Conclusions: The majority of respondents reported using videos to prepare for surgery and YouTube is the preferred source. Posting surgical videos to YouTube may allow for maximal access to learners who are preparing for surgical cases.

 

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