59.08 Skin Fibrosis is Decreased by Local Application of Doxycycline Without Compromising Tensile Strength

Posted on January 18, 2018

R. E. Jones1,2, A. L. Moore2,3, M. P. Murphy2, D. S. Foster2, S. Mascharak2, B. Duoto2,4, D. Irizarry2,5, E. A. Brett2, G. Wernig6, M. T. Longaker2  1University Of Texas Southwestern Medical Center,General Surgery,Dallas, TX, USA 2Stanford University,Department Of Surgery,Palo Alto, CA, USA 3Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA 4San Jose State University,San Jose, CA, USA 5Beth Israel Deaconess Medical Center,Department Of Surgery,Boston, MA, USA 6Stanford University,Department Of Pathology,Palo Alto, CA, USA

Introduction: Cutaneous scarring represents a challenging clinical problem which results in disability, deformity, and psychosocial dysfunction. Matrix metalloproteinase inhibitors are important in regulating scar tissue deposition and extracellular matrix remodeling. Doxycycline is an antibiotic approved by the Federal Drug Administration known to function as a matrix metalloproteinase inhibitor. Given this chemical quality, we theorized that doxycycline may positively affect wound healing. We tested the effect of locally-applied doxycycline on skin fibrosis and bacterial contamination.

Methods: Experiments were conducted with C57BL/J6 mice to test scar thickness, tensile strength, and bacterial colony load in wounds treated with doxycycline or phosphate buffered sodium (PBS). All mice underwent dorsal excisional skin wounding in accordance with previously published work. Doxycycline or PBS was then injected into the base of acute wounds at varying concentrations. Wounds were harvested 15 days postoperatively. Histologic analyses were performed to measure scar thickness and collagen content, including staining with hematoxylin and eosin, trichrome, and picrosirius red. The tensile strength of healed wounds was tested. Wounds were swabbed and cultured, and bacterial colonies were tabulated. Results were compared utilizing the Mann-Whitney U test.

Results: Scar thickness was significantly decreased by 37% in wounds treated with 2 mg/mL doxycycline versus PBS (*p = 0.0107). Doxycycline treated wounds exhibited significantly decreased proportions of mature collagen as compared to control wounds (*p = 0.0470). Bacterial loads were similar at postoperative days 1, 3, 5, 7 and 9 in doxycycline and control mice (p > 0.05). Finally, tensile strength was not significantly different in treatment versus control mice (p = 0.6334).

Conclusions: We show that local application of 2 mg/mL doxycycline decreases scarring in comparison to PBS treated wounds without compromising tensile strength. Additionally, bacterial contamination in treated versus control wounds was not significantly different, suggesting that doxycycline functions as a vulnerary agent to reduce fibrosis in non-antimicrobial manner. Given the longstanding safety profile of doxycycline, this application could be rapidly translated for use in acute injury, postoperatively, hypertrophic scars and keloids, and burns. 

59.06 Macrophage Transplantation Accelerates Repair of Cutaneous Wounds but not Cranial Defects

Posted on January 18, 2018

M. R. Borrelli1, M. S. Hu1, W. Hong1, G. G. Walmsley1, M. P. Murphy1, M. Lopez1, R. C. Ransom1, H. P. Lorenz1, M. T. Longaker1  1Stanford University,Plastic Surgery And Pediatric Regenerative Medicine,Palo Alto, CA, USA

Introduction:
An urgent biomedical need exists to repair both soft tissue and skeletal defects. With a growing incidence of comorbidities such as diabetes, peripheral vascular disease, and obesity, the number of chronic, non-healing wounds is increasing rapidly, costing the United States over $25 billion per year. Additionally, repair and reconstruction of the craniofacial skeleton represents a significant burden with over 50,000 craniotomies/craniectomies per year accounting for $585 million in medical care. We previously demonstrated that the supraphysiologic transplantation of macrophages delivered in a pullalan-collagen composite biomimetic hydrogel scaffold accelerated wound healing in wild type and diabetic mice through a mechanism related to angiogenesis. However, the effect of macrophage transplantation in skeletal defects is yet unknown. Herein, we perform supraphysiologic transplantation of macrophages into cranial defects to assess for improved healing.

Methods:
CD1 athymic nude mice at 60 days of age were anesthetized and unilateral full-thickness critical-size calvarial defects (4 mm in diameter) were created in the non-suture associated parietal bone. Mouse macrophages (1.0 x 10^6 cells), that were isolated from FVB-L2G mice and seeded onto hydroxyapatite-poly(lactic-co-glycolic acid) (HA-PLGA) scaffolds and incubated overnight, were placed into the cranial defects. Micro-CT evaluation was performed at 0, 2, 4, 6, and 8 weeks. Osseous healing was quantified using Adobe Photoshop. Three-dimensional in vivo imaging system (IVIS)/micro-CT was used to determine survival of macrophages in vivo.

Results:
Whereas supraphysiologic transplantation of macrophages accelerated healing of full-thickness cutaneous wounds, macrophage transplantation did not improve healing of critical-size calvarial defects through 8 weeks following creation of defects (p>0.05). This was confirmed with histology. Three dimensional IVIS/micro-CT demonstrated survival of macrophages through 8 weeks.

Conclusion:
Although macrophage transplantation accelerates wound healing via increased angiogenesis, supraphysiologic delivery to critical-size calvarial defects had no effect despite survival of transplanted macrophages. Further research may elucidate the exact mechanism that allows macrophage transplantation to accelerate healing of cutaneous wounds, but not cranial defects.
 

59.07 Ischemia-Reperfusion Injury Induces Neuronal Cell Division in the Murine Enteric Nervous System

Posted on January 18, 2018

C. J. Greig1,2, C. J. Park1, S. J. Armenia1, L. Zhang1, R. A. Cowles1  1Yale University School Of Medicine,New Haven, CT, USA 2University Of Massachusetts Medical School – Baystate Medical Center,Springfield, MA, USA

Introduction:

The enteric nervous system (ENS) has long been considered a static environment, lacking significant neurogenesis under steady-state conditions. Recent evidence suggests, however, that certain stimuli can induce neurogenesis originating from enteric neuronal precursors. While much about this process remains unknown, emerging data suggest the generation of new neurons may occur via two different mechanisms: differentiation from existing neuronal precursors or generation of new mature neurons via cell division. Previous experiments suggested that bowel resection could induce the generation of new mature neurons, but whether this occurred due to cell division was unclear. In the current study, we hypothesized that ischemia-reperfusion injury would induce neuronal precursor cells to generate new neurons via cell division.

Methods:

Wild type (WT) C57Bl/6 controls and Nestin_CreER/eGFP bitransgenic mice expressing eGFP in a tamoxifen-inducible manner and under the control of the actin promoter were used for experiments. Following tamoxifen induction, bitransgenic mice underwent ischemia-reperfusion (IR) injury via superior mesenteric artery occlusion (T=30 and 45 min). WT and bitransgenic controls received tamoxifen injections alone without IR, and bitransgenic sham laparotomy animals received the same surgical procedure as IR animals without SMA clamping. IR animals received injections of EdU at the start of the reperfusion period and again at day 5. After the reperfusion period (3 or 10 days), distal ileal segments were obtained from all animals. Whole mounts of the myenteric and submucosal plexuses were created by mechanically peeling these layers after fixation. Peeled specimens were then stained with fluorescent antibodies targeting a marker of mature neurons, HuC/D, and GFP. A click chemistry assay was used to detect Edu in the whole mount specimens.

Results:

WT and bitransgenic controls demonstrated no evidence of GFP-expressing mature neurons, suggesting no demonstrable neurogenesis occurred at baseline. Bitransgenic sham laparotomy mice demonstrated a low incidence of neurogenesis, as evidenced by mature neurons expressing GFP in one of four high-powered fields. In IR animals, we found evidence of extensive neurogenesis with GFP-expressing mature neurons in nearly all high-powered fields. Moreover, EdU was detected in most mature, GFP-expressing neurons, suggesting that these neurons had been generated from precursors via cell division.

Conclusion:

Ischemia-reperfusion injury appears to induce a substantial mitotic response in the ENS of the murine ileum, and this process appears to occur via cell division of neuronal precursors. Interestingly, anesthesia and laparotomy alone appear to represent a sufficient stimulus to induce neurogenesis, but to a lesser extent than ischemia-reperfusion. While preliminary in nature, these data provide insight for future study into the mechanisms of enteric neurogenesis. 

59.04 Adipose Derived Stromal Cell Enhancement of Cancer Growth Likely Due To Specific Gene Upregulations

Posted on January 18, 2018

D. M. Irizarry1, J. S. Flacco1, C. P. Blackshear1, C. F. Montenegro1, D. Nguyen1, N. Quarto1, A. Giaccia1, M. T. Longaker1,2, D. C. Wan1  1Stanford University,Plastic And Reconstructive Surgery,Palo Alto, CA, USA 2Institute For Stem Cell Research And Regenerative Medicine,Palo Alto, CA, USA

Introduction: Many oncologic surgeries create significant defects with a need for reconstruction. Further, radiation therapy induces fibrotic damage to skin. Fat grafting has been utilized to address these soft tissue deficiencies, and studies have suggested a regenerative effect on radiation-damaged skin. Inclusion of supplemental mesenchymal cells to fat grafts have also been shown to augment these effects. However, mesenchymal cells have been described in animal models to promote carcinoma growth, and with head and neck cancer, may enhance tumor invasion and formation of micrometastases. As cancer of the oral cavity is among the most common types of head and neck cancer, we chose to evaluate the effects of ASCs on the SAS HNSCC cell line.

Methods: SAS HNSCC cells were plated with either regular or ASC conditioned media. They were lifted and counted every other day for 6 days. For the in-vivo arm, 5X105 GFP labeled SAS HNSCC cells were injected into the subcutaneous plane of the scalp of mice either alone, in addition to a 200uL fat graft, or in addition to a 200uL fat graft and 10,000 ASCs. CT scans were taken weekly for 4 weeks. To examine what factors released by ASCs might be responsible for promoting cancer proliferation, differential protein component analysis was performed on conditioned media from co-cultured ASCs and cancer cells and compared to media from cancer cells alone and ASCs alone.

Results: Cancer cells grown in ASC conditioned media show greater proliferation than those grown in regular media. At the 3 and 4 week timepoints, there was significantly greater tumor volume by microCT in mice receiving cancer with fat graft and ASCs compared to mice receiving cancer alone or in addition to a fat graft. Multiple upregulated factors were identified in the ASC-cancer co-culture medium when compared to cancer alone or ASCs alone, most notably epidermal growth factor was upregulated 1.4-fold, hepatocyte growth factor was upregulated 1.9-fold, and monocyte chemotactic protein was upregulated 5.0-fold. Interestingly, each of these three factors have been implicated in growth and/or metastatic development in head and neck cancer.

Conclusion: While the risk for head and neck cancer recurrence following fat grafting with or without ASC enrichment remains unclear, our preliminary data have shown that ASCs potentially stimulate cancer cell proliferation and tumor formation, and that this may be secondary to one or more likely multiple of the factors we have identified in our component screen. Our ongoing studies are currently testing this hypothesis. Although ASCs and fat grafting may have enormous regenerative potential, the oncologic ramifications of their use must therefore be carefully considered before widespread application.

59.05 Reverse Thermal Gel: Our Candidate for an Early, Minimally Invasive Method to Patch Myelomeningocele

Posted on January 18, 2018

J. R. Bardill1, D. Park1, U. Shabeka3, A. I. Marwan1,2,3  3University Of Colorado Denver,Laboratory For Fetal Regenerative Biology,Aurora, CO, USA 1University Of Colorado Denver,Bioengineering,Aurora, CO, USA 2Children’s Hospital Colorado,Pediatric Surgery,Aurora, CO, USA

Introduction:  Myelomeningocele (MMC) results in lifelong neurological and functional deficits.  In-utero repair closes the defect, resulting in a 50% reduction in post-natal ventriculoperitoneal shunting. However, it is associated with maternal and fetal risks.  Novel fetoscopic approaches using 2-3 ports, pre-fabricated patches, and skin closure demonstrate promising results, however, they are performed late and have a prolonged operative time. Our novel, Reverse Thermal Gel (RTG) has posed a unique solution for an early, minimally invasive delivery of a patch.  So far, we have shown fast in-situ gelling properties and RTG stability after long term exposure in amniotic fluid. Moreover, RTG is non-toxic to cells and embryos in culture. Here, we hypothesize that RTG can serve as an in-vitro 3D cellular scaffold for mouse skin fibroblasts and will be successfully injected and localized to the MMC defect of mouse embryos with minimal inflammatory response.

Methods:  1. In-vitro 3D-RTG cellular scaffold:  Mouse skin fibroblasts were mixed with RTG-media solution, followed by gelling at 37°C to encapsulate the cells within the RTG.  The fibroblasts were cultured for 3 days and then viability was assessed using a live/dead stain and confocal microscopy.  2. RTG injection into mouse MMC defect embryos:  Grainyhead like-3 (Grhl3) mice express MMC phenotypes.  Pregnant E16.5 Grhl3 mice underwent laparotomy to expose uterine horns.  A RTG solution or saline was applied onto the MMC defect by transuterine injection.  On E19, the embryos were harvested to assess RTG coverage of the MMC defect.  3. Inflammatory response to RTG injection:  Defect tissue was stained for CD68 and F4/80 macrophage antibodies and imaged on a fluorescent microscope.

Results:  Fibroblast 3D-RTG culture live/dead staining reveals 90-95% live cells after 3 days. The RTG successfully applies to mouse MMC defects and forms a stable gel covering the defect.  A total of 6 MMC defect embryos are recovered at E19 harvest:  3 defect embryos demonstrate more than 50% coverage of the defect, while the other 3 show less than 50% coverage.  CD68 and F4/80 macrophage response demonstrates little to no difference in inflammation comparing RTG and saline injections in defect embryos.

Conclusion:  Using a mouse model, we demonstrate that by simple needle injection, a RTG can cover a mouse MMC defect and remain at the site of the defect until harvest with minimal inflammatory response.  However, RTG defect coverage decreases over time.  Moreover, the RTG has in-vitro­ fibroblast scaffolding properties, which could allow for native cellular integration into the patch and/or allow cellular treatments to be incorporated into the patch. Based on our results, we are currently modifying the RTG to enhance the adhesive properties. Overall, our work demonstrates that a RTG is a promising candidate for a minimally invasive approach to patch MMC and will be conducive to transitioning to a fetoscopic approach in a sheep model.

59.02 Rapamycin Inhibits Primary and Recurrent Heterotopic Ossification in Genetically Susceptible FOP Mice

Posted on January 18, 2018

C. Hwang1, S. Ucer1, C. Pagani1, N. Patel1, A. Vaishampayan1, M. Sorkin1, M. T. Chung1, J. Li1, C. Breuler1, C. Priest1, A. N. Economides3, S. Agarwal1, Y. Mishina2, B. Levi1  1University Of Michigan,Section Of Plastic Surgery,Ann Arbor, MI, USA 2University Of Michigan,Department Of Biologic And Material Sciences, School Of Dentistry,Ann Arbor, MI, USA 3Regeneron Pharmaceuticals And Genetics Center,Tarrytown, NY, USA

Introduction: Heterotopic ossification (HO) is a debilitating formation of ectopic bone restricting joint mobility and causing chronic pain. Fibrodysplasia Ossificans Progressiva (FOP) is a congenital variant of HO caused by a genetic mutation in a bone morphogenetic receptor that causes severe, progressive lesions resulting in immobility and fatal mechanical respiratory failure at a premature age. With increased proclivity for osteogenesis at baseline, surgical extirpation is contraindicated in FOP patients due to universal recurrence. This disease presents a unique clinical challenge with no current treatments available. Previous research has shown mTOR inhibition by rapamycin to be a viable therapy for reducing trauma-induced HO. Mice of an FOP mouse model with a hyperactivating mutation in the ACVR1 receptor (ACVR1R206H/+) were examined to determine the effects of rapamycin on both primary and post-surgical HO.

Methods: Mice were designated for 2 distinct study arms. In the primary HO cohort, ACVR1R206H/+ P21 mice received bilateral hindlimb cardiotoxin (CTX) with Ad.cre injection and stratification to daily i.p. rapamycin (n=10 hindlimbs) v. PBS control (n=12). 3 weeks later, mice were scanned with in vivo µCT. Mice within the separate recurrence cohort received initial Ad.cre/CTX followed by baseline CT scans, Ad.cre reinjection bilaterally, and surgical removal of HO at the 3 week timepoint. After surgery, mice were randomized to daily rapamycin (5 mg/kg, n=12) or PBS (n=8) for 3 weeks with endpoint µCT. Contours were drawn manually around HO to compute total volumes at 800HU. Primary and recurrence cohorts were analyzed by Student’s t test and log-transform/ANOVA/Hochberg post-hoc respectively.

Results: Mice treated with rapamycin for 21 days showed 261-fold less ectopic bone when compared to PBS vehicle injection (p=0.002). In post-surgical mice, PBS injection showed statistically similar HO volumes to baseline volumes of pre-excision mice (p=0.054). However, rapamycin treatment reduced the recurrence of HO volume 11-fold (p=.044, Figure). Geometric means from de-transformed data for pre-excision v. PBS, pre-excision v. rapamycin, and PBS v rapamycin were 0.061, 1.398, and 22.961 respectively.

Conclusions: These studies demonstrate that rapamycin prevents primary development of HO and is also effective in preventing recurrence following surgical excision in a FOP mouse model. This study further corroborates rapamycin as a promising candidate for primary and post-surgical HO prophylaxis in children with FOP. Existing literature implicates similar molecular mechanisms among various etiologies of HO, suggesting a putative role for rapamycin even beyond FOP in post-traumatic and post-surgical HO patients.

59.03 Loss of Intestinal Alkaline Phosphatase Leads to Distinct Changes in Bone Phenotype

Posted on January 18, 2018

F. Kuehn1, F. Adiliaghdam1, S. R. Hamarneh1, R. Vasan1, E. Liu1, Y. Liu1, J. M. Ramirez1, F. C. Ko2, M. L. Bouxsein2, M. N. Wein2, M. B. Demay2, R. A. Hodin1  1Massachusetts General Hospital,Surgery,Boston, MA, USA 2Massachusetts General Hospital,Endocrinology,Boston, MA, USA

Introduction:  The gut is becoming increasingly recognized as the source of various systemic diseases, and recently it has been linked to bone metabolism and formation via the so-called gut-bone axis. The microbiome and gut-derived mediators such as lipopolysaccharides (LPS) are thought to impact bone metabolism and administration of beneficial bacteria (probiotics) such as lactobacillus has been shown to result in higher bone mineralization and bone strength. The gut brush border enzyme intestinal alkaline phosphatase (IAP) plays an important role in controlling calcium absorption, inhibiting LPS and other inflammatory mediators responsible for endotoxemia, and, also appears to preserve the normal of gut microbiota. Interestingly, IAP-deficient mice (AKP3-/-) also display a significant decrease in Lactobacillus, the genus shown to be beneficial to bone. Based on this background, we sought to determine the impact of IAP-deficiency on bone formation in mice.

Methods:  Transcript levels of IAP in mouse bone were measured using qPCR. Femurs of young (3 months) and old (21 months) IAP-KO and WT mice were analyzed by micro-CT scan and histopathology. Serum levels of alkaline phosphatase (AP) as a marker of active bone formation, and, serum calcium and phosphorus levels were measured in IAP-KO and WT mice. In-vitro target cell response upon exposure to blood serum from IAP-KO and WT mice was measured using primary bone marrow macrophages.

Results: IAP was not expressed in bone of WT mice. Transcript levels were similar to IAP-KO counterparts (IAP-WT vs. IAP-KO, 56.9 ± 17.7 vs. 41.4 ± 21.3 Relative Expression, p=0.4) and vanishingly low when compared to duodenum expression levels (bone vs. duodenum, 56.9 ± 17.7 vs. 25,430.3 ± 10,884.5 Relative Expression, p=0.01). Histological examination of 3-month old IAP-KO and WT did not show any differences, whereas older IAP-deficient mice showed a very distinct bone phenotype in histology and CT-scan (image 1). Younger KO mice did not display any abnormal levels in blood chemistry analysis but older IAP-KO animals showed an isolated increase in AP serum levels, significantly higher than in control animals (IAP-WT vs. IAP-KO, 80 ± 27.4 U/I vs. 453 ± 107.5 U/I, p =0.004). Normal GGT levels ruled out an underlying hepatic pathology causing the AP increase. Finally, to determine to the extent that systemic serum may contribute to inflammation in peripheral organs such as the bone marrow we tested the inflammatory response of target cells exposed to systemic blood serum from IAP-KO and WT mice. Serum from IAP-KO mice induced a significantly higher inflammatory target cell response than serum derived from WT counterparts (p<0.01).

Conclusion: Through its multiple functions, IAP seems to play a crucial role in connecting the gut to the bone. IAP-deficiency leads to chronic changes in bone formation, probably through dysbiosis and dissemination of pro-inflammatory mediators. 

58.10 Blue Light Improves Survival After Pneumonia By Augmenting Circadian Protein Expression

Posted on January 18, 2018

J. E. Griepentrog1, A. J. Lewis1, X. Zhang1, J. S. Lee2, M. R. Rosengart1  2University Of Pittsburgh,Department Of Medicine, Division Of Pulmonary, Allergy And Critical Care Medicine,Pittsburgh, PA, USA 1University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA

Introduction:

Pneumonia is a major cause of sepsis and acute lung injury, with three million cases reported annually in the US; it is the sixth most common cause of death. The causal mechanisms of acute alveolar damage include an excessive accumulation of neutrophils in the lung and the subsequent production of inflammatory mediators and neutrophil-mediated oxidant damage. Light has been shown to influence an organism’s response to stress. Circadian proteins, including REV-ERB alpha, are known to attenuate inflammation and regulate mitochondrial biogenesis, and modulating them may enable us to modify the response to sepsis. Our previous animal work demonstrated that blue light enhances bacterial clearance and improves survival during pneumonia. However, the cellular and neurophysiologic mechanisms by which blue light harnesses circadian biology to alter macrophage phagocytic function are unknown.

Methods:
Male C57BL/6 mice were intratracheally administered Klebsiella pneumoniae (6000 CFUs) and immediately exposed to an initial 36 hours followed by 12 hours daily of bright (1400 lux) blue (442nm) or red (617nm), or ambient white fluorescent (400 lux) light. Some mice underwent splenectomy or exposure to the α7 nicotinic AChR antagonist, alpha-bungarotoxin. After 72 hours, mice were euthanized, the lungs lavaged, and the blood/organs harvested. Tissue and blood were analyzed for bacterial colony forming units (CFUs). Lung, spleen, and alveolar macrophages expression of REV-ERB alpha was analyzed by immunofluorescence and immunoblot. Cytokine and BAL protein concentrations were quantified by ELISA and BCA. Statistical analysis was performed by Wilcoxon rank-sum tests at α = 0.05.

Results:
By contrast to either red or ambient white light, blue light reduced lung tissue bacteria (p = 0.06), bacteremia (p = 0.10), and bacterial dissemination into tissues and improved survival (log-rank p = 0.02). Blue light attenuated alveolar neutrophil influx (p = 0.0001), yet preserved the alveolar macrophage population. Either inhibiting cholinergic tone with alpha-bungarotoxin or splenectomy eliminated the protective effects of blue light: Blue vs. Blue with bungarotoxin, lung CFUs (p = 0.004), blood CFUs (p = 0.02), neutrophil influx (p = 0.002). Lung tissue and alveolar macrophage expression of REV-ERB alpha was increased after blue light.

Conclusion:
Blue light functions through a cholinergic pathway and the spleen to augment lung innate immunity and bacterial clearance, while reducing neutrophilic inflammation. The mechanisms appear to involve an augmentation in REV-ERB alpha expression in alveolar macrophages and lung tissue. Further studies are required to determine how altering REV-ERB alpha affects macrophage phagocytic function.
 

59.01 Differences in vascular reactivity of arteries of colon tumors vs. non tumoral arteries in humans: role of phenylephrine, thromboxane and bradykinin.

Posted on January 18, 2018

N. Cupitra1, A. Múnera Duque2, JP León2, R. Narvaez-Sanchez1  1PHYSIS Group, Faculty of Medicine, University of Antioquia, Medellín, Antioquia, Colombia 2Department of Surgery, Faculty of Medicine, University of Antioquia, Medellín, Antioquia, Colombia

Cancer is one of the leading causes of death on the planet: in 2012, 8.2 million deaths were attributed to it, with the highest number of victims caused by lung cancer (19, 4%), hepatic (9.1%), gastric (8.8%) and colorectal (8.4%); (1). As an abnormal growth of cells, cancer needs vascular changes for growing and metastases. In the last two decades the tumor vascular system and its possible role in the development of cancer has been investigated, proposing it as a target for the control of nutrient supply, by modification of vascular tone, angiogenesis and neovascularization (2). Vascular reactivity (VR) could be a limiting factor for growth in cancer, since growing tissues require vasodilation and new vascularization. Promoting vasoconstriction in the tumor could reduce the supply of nutrients, while promoting vasodilation could increase therapies to reach more cancer cells. The present work aims to quantify differences in human VR of tumoral (TU, arteries which directly irrigate the tumor), extratumoral (ET, arteries of a tumoral patient, which do not irrigate tumor but the distal and proximal colon near the tumor) and non-tumoral arteries (NT, branches of mesenteric circulation of colectomized patients due to a non tumoral pathology), to the stimulus of potassium chloride (KCl), phenylephrine (FE), ET-1, U-46619 (analog of thromboxane A2), and bradykinin (BK).

Six patients with colon cancer and two with non tumoral pathology, from 30 to 80 years old, underwent hemicolectomy at “IPS Universitaria Leon XIII” Hospital, in Medellin, Colombia. All patients signed the respective informed consent. Mesenteric branches were collected and preserved in Krebs-Henseleit solution (SK, composition (mEq/liter): Na+, 143.5, K +, 5.4, Ca2 +, 5.1, Mg2+, 2.4, Cl-, 128, H2PO4-, 1.2, HCO3, 24.9, SO42-, 2.4, and glucose, 10) at 4° C where excess of peripheral tissue was removed. The arterial segment was cut into rings 3-5 mm long, which were mounted on stainless steel hooks in parallel through the lumen. One of the hooks was attached to a force transducer (ADInstruments Ltd, Model MLT0201, UK), and the other was held as a fixed support. The preparation was mounted in a 10 ml organ bath (Panlab/ Harvard, Model LE01046, Spain) containing SK. The tissue was maintained at 37° C and bubbled with carbogen (95% O2- 5% CO2). Each preparation was stabilized at 2 grams of basal tension along 90 minutes, before initiating stimulations with agonists, changing SK every 20 minutes. After stabilization the arterial ring was stimulated twice with KCl 4×10-2 M recording the response for 10 minutes, and washing for 20 minutes between stimuli. Then U-46619 1×10-7 M and BK 1×10-5 M were applied to confirm the presence of endothelium. This verification was performed on all arterial rings. Then the evaluation was done applying in some rings KCl at a single dose KCl 4×10-2 M, or FE at cumulative dose of 5×10-8 M to 1×10-5 M, or U-46619 cumulative dose of 1×10-13 M to 1×10-7 M, or, precontracting with U-46619, BK at cumulative dose of 1×10-9 M to 1×10-5 M. The maximal relative response (“Emax%KCl”, maximal contraction normalized in respect to maximal contraction to KCl) and sensitivity (EC50, concentration which generates 50% of response) were recorded by the Powerlab PL-3504 polygraph (ADInstruments, Australia). Analyzes were performed with t-Student and ANOVA with significance level p <0.05. The EC50 was calculated by non-linear regression of the concentration-response curves. The statistical package GraphPad prism 6 Statistics was used. Experimental results are presented as means ± SEM of the Emax and EC50:

The greater response of TU but not of NT to KCl suggests histological differences that we have not specified yet. The increase in Emax to FE and EC50 U-46619 of TU and ET with respect to NT, suggests that the tumor microenvironment favors the contractile response in the arteries that irrigate it: it has been reported that oxidative stress activates kinases that promote proliferation and resistance to anticancer agents, and induces endothelial dysfunction that worsens vasodilation. The beta-adrenergic receptors participate in the initiation and progression of the tumor; but more evidence is lacking about the role of alpha-adrenergic signaling and thromboxane A2 in vascular control in cancer. The higher sensitivity to U-46619 in TU and ET suggests an increase in the activity of the thromboxane-A2 dependent mechanisms, coinciding with what is reported in greater activation of thromboxane-prostanoid receptors. Our relaxation analysis did not show differences of VR to BK in Emax nor in EC50 between TU and ET, contradicting a possible endothelial damage, at least in TU and ET. These preliminary results require contrasting with non-tumoral arteries, measuring differential expression of receptors, doing histology evaluating integrity of the vascular wall, and quantifying probable endothelial dysfunction by specifying affected mechanisms, using specific blockers.

58.09 Traumatic Endotheliopathy is Associated With Arrival Hypocoagulability in Level-1 Trauma Patients

Posted on January 18, 2018

E. Gonzalez Rodriguez1, J. Cardenas1, B. Cotton1, Y. Wang1, S. Ostrowski2, J. Stensballe2,3, P. Johansson1,2, J. Holcomb1, C. Wade1  1University Of Texas Health Science Center At Houston,Center For Translational Injury Research,Houston, TX, USA 2Copenhagen University Hospital,Section For Transfusion Medicine, Capital Region Blood Bank,Copenhagen, -, Denmark 3Copenhagen University Hospital,Department Of Anesthesia, Centre Of Head And Orthopedics,Copenhagen, , Denmark

Introduction: Traumatic endotheliopathy (EOT) is a syndrome that arises from breakdown of the glycocalyx. This results in shedding of syndecan-1 and endothelial activation, which triggers downstream effects that lead to coagulation disturbances, leakage, organ failure and poor outcomes. Because the hemostatic potential of patients with EOT is unclear, we aimed at characterizing it in this study. We hypothesized that the presence of EOT is associated with acute coagulation derangements as assessed by rapid thrombelastography (r-TEG) that are not found in syndrome-free patients.

Methods: To test our hypothesis, we conducted a prospective observational study in trauma patients with the highest level of activation. Demographic, clinical and laboratory data, injury severity (ISS) scores, and transfusion data were collected. Upon hospital arrival, blood samples were obtained to measure i) Syndecan-1 and soluble thrombomodulin (sTM) (from plasma), ii) hemostatic potential measured by r-TEG. Clinically meaningful r-TEG values are the activated clotting time (ACT), values over 128 seconds indicate factor deficiency or severe hemodilution; α-angle (abnormal < 56°) and maximal amplitude (MA) (hypocoagulable < 55; hypercoagulable > 72 mm), both altered in platelet dysfunction or hypofibrinogenemia. LY30 elevations (> 3.0%) reflect hyperfibrinolysis. Based on a previous study, patients were dichotomized into an EOT+ group (syndecan-1≥ 40 ng/ml) or EOT- group (< 40 ng/ml). Differences between groups were assessed with non-parametric tests.

Results:of the 527 that met inclusion criteria, 36% (n=189) had EOT+. Demographic data were comparable between groups. In general, patients with EOT+ had higher ISS (median EoT+ 24 vs.17 EoT-), but the frequency of TBI was similar in both groups. EOT+ patients had more endothelial activation/injury than syndrome-free patients indicated by higher sTM median levels (7.2 vs. 4.9 ng/ml; p<0.05). All r-TEG parameters, except LY30 were significantly different between groups. In contrast to EOT- patients, a higher proportion of EOT+ patients had prolonged ACT, and lower MA, α- angle values, and platelet counts (< 150 10 3 µl) than the EOT- group (Figure). These alterations reflected a hypocoagulable state in EOT+ patients. EOT+ patients required blood transfusions more frequently than EOT- patients (median 4-hour total blood units was 4 in EOT+ vs. 0 in EOT-; p<0.05). 

Conclusion:EOT is a syndrome associated with a state of r-TEG hypocoagulability, and an increased need for early transfusion of blood products. This is likely driven by exposure and activation of the endothelium after glycocalyx breakdown triggered by sympathoadrenal activation after trauma.

 

58.06 Early Continuous Renal Replacement Therapy Alleviates Post-Cardiac Arrest Syndrome after Hemorrhage

Posted on January 18, 2018

P. Shen1,2,3, J. Xu1,2,4, S. Xia1,2, S. Liu1,2, Z. Li4, M. Zhang1,2  1Department Of Emergency Medicine,Second Affiliated Hospital, Zhejiang University School Of Medicine,Hangzhou, ZHEJIANG, China 2Institute Of Emergency Medicine,Zhejiang University,Hangzhou, ZHEJIANG, China 3Department Of Intensive Care Medicine,The First Hospital Of Jiaxing,Jiaxing, ZHEJIANG, China 4Department Of Emergency Medicine,Yuyao People’s Hospital, Medical School Of Ningbo University,Ningbo, ZHEJIANG, China

Introduction: The poor outcome of traumatic cardiac arrest is partly attributed to post-resuscitation systemic inflammation and multiple organ injury. Continuous renal replacement therapy (CRRT) is used for acute renal failure following resuscitation, however, the optimal timing of CRRT initiation might be controversial. In this study, we investigated the effects of CRRT early initiated after resuscitation on post-cardiac arrest syndrome following severe hemorrhage. We hypothesized that early initiation of CRRT would alleviate post-resuscitation hemodynamic abnormality, systemic inflammation, cardiac and cerebral injuries in a porcine model of hemorrhage-induced traumatic cardiac arrest.

Methods: Sixteen male domestic swines weighing between 28-33 kg were utilized. Forty percent of estimated blood volume was removed over an interval of 20 minutes. Subsequently, ventricular fibrillation was electrically induced and untreated for 5 minutes while defibrillation was attempted after 5 minutes of cardiopulmonary resuscitation. The resuscitated animals were randomized to receive either CRRT (n=7) or sham control (n=9). At 5 minutes after successful resuscitation, CRRT was performed with the mode of continuous venovenous hemofiltration for a total of 6 hours. All animals were observed for 24 hours. Continuous variables were presented as mean ± SD and compared with one way analysis of variance.

Results: After resuscitation, mean arterial pressure was significantly increased and heart rate was significantly decreased in the CRRT group when compared with the control group (6-hr mean arterial pressure, 129±4 vs. 93±9 mmHg, P<0.001; 6-hr heart rate, 116±16 vs. 172±34 beats/min, P=0.003). Post-resuscitation cardiac and neurologic dysfunction were significantly improved in the CRRT group compared to the control group (6-hr ejection fraction, 67±6 vs. 59±5 %, P=0.015; 24-hr neurological deficit score, 95±13 vs. 190±31, P<0.001). Serum troponin I at 6 hours ( 346±28 vs. 433±46 pg/ml, P=0.003) and serum neuron specific enolase at 24 hours (15.9±1.9 vs. 28.5±1.1 ng/ml, P<0.001) were also significantly reduced in the CRRT group. Additionally, significantly lower serum tumor necrosis factor-α (570±71 vs. 1031±39 pg/ml, P<0.001) and interleukin-6 (224±29 vs. 416±16 pg/ml, P<0.001) at 24 hours after resuscitation were achieved with the CRRT compared to the control group.

Conclusion: Early initiation of CRRT significantly improved cardiac and neurological outcomes after resuscitation while alleviated systemic inflammatory response in a porcine model of traumatic cardiac arrest.

 

58.07 Post-Injury Righting Time Paradoxically Decreases with Cumulative Mild Concussive Events in Rodents

Posted on January 18, 2018

M. Srour1, G. Barmparas1, N. K. Dhillon1, N. Cho1, M. Alkaslasi1, E. J. Smith1, E. J. Ley1, G. M. Thomsen1  1Cedars-Sinai Medical Center,Los Angeles, CA, USA

Introduction:  NFL athletes sustain acute head trauma with no perceived motor deficit although sidelines analysis appears to indicate altered mental status. It has been hypothesized that repeat mild traumatic brain injury (TBI) induces tolerance that may lead to quicker recovery, although this has not been definitively demonstrated clinically or in vivo.  We studied the time to regain consciousness following mild TBI in a rodent model to better understand how repeat injury alters the acute recovery.

Methods:  Righting time for 44 wild type (WT) rats and 17 WT mice were examined over a 5-week period. Rodents were administered repeat, bilateral, closed skull TBI with a controlled cortical impact device using varying injury parameters to deliver mild or moderate concussion. Starting at postnatal day 60, rats were administered either once weekly moderate injury (n=16 TBI, 16 sham) or 3x per week mild injury (n=7 TBI, 5 sham). Mice (aged >360 days) were administered mild repeat injury (n=11 TBI, 6 sham) once per week for 5 weeks. Righting time, defined as the length to turn over from supine to prone on all four paws after removal from anesthesia, was assessed following TBI or sham procedure.

Results: Rats administered a mild, 3x per week, concussion exhibited a delay in righting time after the first week of injuries that significantly decreased over time (Figure). Similarly, in mice that received a mild concussion once per week for 5 weeks, righting time significantly decreased over time (not shown). Rats that received a more moderate concussion once per week for 5 weeks demonstrated significantly longer righting times relative to sham that did not decrease over time (not shown).

Conclusion: Although cumulative incidence of mild TBI may lead to permanent long-term deficits in motor and balance, the acute recovery after each episode may unexpectedly decrease.  Clinically our findings suggest that the expected symptoms after repeat blows to the head may diminish over time, which may complicate both the concussion diagnosis and prevention of long-term damage.

 

58.04 Human Mesencymal Stem Cells Hasten the Kinetics of Clot Formation

Posted on January 18, 2018

M. J. George1, K. Prabhakara1, N. E. Toledano-Furman1, Y. Wang1, S. D. Olson1, B. S. Gill1, C. E. Wade1, C. S. Cox1  1University Of Texas Health Science Center At Houston,Pediatric Surgery,Houston, TX, USA

Introduction:  Human mesenchymal stem cells (MSCs) have shown efficacy in reducing inflammation after trauma. Previous studies using bone marrow derived MSCs to treat traumatic brain injury (TBI) demonstrate downregulation of inflammatory biomarkers and preservation of central nervous system architecture. However, MSCs express tissue factor (TF) which stimulates coagulation. We hypothesize that MSCs exhibit procoagulant activity linked to TF expression. We evaluated pro-coagulant activity and tissue factor expression of five different tissue sources of MSCs from multiple donors. 

Methods:  Multiple MSC samples from bone marrow, adipose, amniotic fluid, umbilical cord, and bone marrow derived cell donors were tested. TF expression and phenotype were quantified using multi-parametric flow cytometry. Fluorescence-activated cell sorting (FACS) separated select samples into high and low tissue factor expressing populations to isolate TF as a test variable. Pro-coagulant activity of the MSCs was measured in-vitro using blood from healthy donors in thromboelastography (TEG) and pooled plasma in calibrated automated thrombogram (CAT). MSCs were tested at concentrations of 10^5 cells/mL, similar to clinical dosing.

Results: All MSC tissue types express pro-coagulant activity that directly correlates with expression of tissue factor. Specifically, time to clot formation measured by R time in TEG decreased logarithmically as percent cells expressing TF increased compared to untreated controls. A Pearson’s product-moment correlation demonstrated strong correlation between percent of control R time and cells expressing TF (r = 0.683, p < 0.0001) (Figure 1). Similarly, time to thrombin generation in CAT decreased with increasing TF expression. High TF expressing MSCs decrease R time more than low TF expressing MSCs when sorted from single samples using FACS. 

Conclusion: Human MSCs demonstrate pro-coagulant activity related to TF expression and hasten the kinetics of clot formation. This effect was proven to be related to TF by demonstrating high TF expressing MSCs decrease TEG R time more than low TF expressing MSCs when sorted from the same sample. 

 

58.05 The Endocannabinoid System: A Biomarker for Pain in the Traumatically Injured?

Posted on January 18, 2018

C. M. Trevino1, S. Chesney2, C. Hillard1, T. DeRoon-Cassini1  1Medical College Of Wisconsin,Surgery Trauma And Acute Care Surgery,Milwaukee, WI, USA 2Marquette University,Milwaukee, WI, USA

Introduction:
Little is known about the progression of acute pain to chronic pain in the traumatically injured population and the risk factors associated with this transition. In addition, there are few reliable or clinically significant biomarkers, for either of these conditions. Preclinical research suggests a role for the endocannabinoid signaling system (ECSS) in pain. Specifically, increased endocannabinoid (EC) signaling that is promoted by tissue injury and contributes at first to reduced pain could also contribute to some forms of pain sensitization and, thus contribute to the development of chronic pain.  We hypothesized that EC levels would correlate with pain scores at baseline and 6 months after traumatic injury. 

Methods:
A prospective longitudinal cohort study was conducted with participants admitted to an Adult Level I Trauma Center post traumatic injury. Participant demographic and injury-related data, pain scores, pain interference scores, and symptoms of Post-Traumatic Stress Disorder (PTSD) were assessed via measures obtained at the time of hospitalization (baseline) and 6 months following the index traumatic event. Non-fasting whole blood samples measuring ECs [2-arachidonoylglycerol (2AG) and anandamide (AEA)] and EC genetic polymorphisms of fatty acid amide hydrolase (FAAH) expression were drawn at baseline and at 6 month follow up. Pearson correlations and ANOVAs were used to determine relationships.    

Results:
280 participants comprised a subset of a population base sample (72.5% Male, 27.5% Female) with 170 participants (61% retention) completing 6-month follow-up. 2AG at baseline was significantly correlated with pain interference at 6 months (r=0.197, p=0.01). AEA at 6 months was significantly correlated with both pain severity (r=0.164, p=0.04) and pain interference (r=0.191, p=0.02) at 6 months. Participants with the rare genetic allele for FAAH had significantly higher 6 month pain severity (likelihood ratio=10.431; p=.034, Cramer’s V=0.175). Pain scores and PTSD here highly correlated both at baseline and at 6 months (r=0.3, p<0.001; r=0.4, p<0.001). 

Conclusion:
The ECSS is known to play a role in both the perception of pain and psychological distress. Similar to previous studies, pain and PTSD was highly comorbid in this population. These data suggest higher levels of 2AG and AEA at 6 months may play a role in the development of chronic pain. Those with genetic FAAH over expression, and ultimately higher ECs, can prevent the decrease in AEA. This decrease in AEA overtime is protective in reducing the activation of ascending pain pathways, thereby potentially affecting the negative feedback of AEA in the development of chronic pain. From these data a significant relationship exists between the EC system and chronic pain. Therefore, the EC system may play a pivotal role in the development of chronic pain from acute traumatic injury and is potentially a target for intervention. 

58.01 Invariant Natural Killer-T-cells mediate sepsis survival via modulation of the Ang-1/Ang-2 pathway

Posted on January 18, 2018

D. S. Heffernan1, T. T. Chun1, C. Chung1, A. Ayala1, J. Lomas-Neira1  1Brown University School Of Medicine,Surgical Research,Providence, RI, USA

Introduction:
We have previously demonstrated a central role for invariant Natural Killer T-cells (iNKT-cells) in modulating the immune response to, and mortality from, intra-abdominal sepsis. Across a spectrum of diseases iNKT-cells can affect the neutrophil response. End organ pulmonary dysfunction, is well recognized as contributing to sepsis related morbidity and mortality. The Angiopoietin (Ang)-1 and Ang-2 pathways are central to the pulmonary vascular endothelial cell response to injury, mediated via neutrophil response and gap junction integrity. Ang-1 promotes anti-inflammatory, pro-survival and anti-permeability reponses, whereas Ang-2 signaling promotes vessel destabilization, vessel leakage and inflammation. Given this, we hypothesize that iNKT-cells mediate their sepsis survival effect via direct/indirect actions on the Ang-1/Ang-2 pathway.

Methods:
Sepsis, induced by Cecal Ligation and Puncture(CLP), versus sham laparotomy, in 8-12 week old C57BL/6 wild type(WT) and iNKT-knockout (iNKT-/-) mice. 24 hours later, blood was collected to measure oxygenation (PaO2/FiO2) and lungs were harvested. Histology, cytokine and Western blot analysis was undertaken for Ang-1/Ang-2, their ligands (Tie-1/Tie-2) as well as Vinculin (marker of endothelial integrity).

Results:
Sepsis did not induce gross histological damage in either WT or iNKT-/- mouse lungs. Further, there was no difference in oxygenation levels (PaO2/FiO2) following sepsis between WT and iNKT-/- mice. However, although sepsis induced a minimal pulmonary neutrophil influx in WT mice, within the iNKT-/- septic mice, a marked influx of neutrophils was noted. Whereas sepsis in WT was associated with increases in KC and MIP-2 levels, no changes were noted in iNKT-/- mice. Within WT, sepsis induced a decline in the pro-survival, anti-inflammatory Ang-1 levels, whereas in iNKT-/- mice, no change in Ang-1 expression was noted. Sepsis was not associated with any change in Ang-2 expression within either WT or iNKT-/- mice. With respect to Angiopoietin receptors, within WT mice sepsis induced a marked increase in Tie-1 and a marked decrease in Tie-2 expression. However, within iNKT-/- mice there was no change in either Tie-1 or Tie-2 expression. Regarding vinculin, as a marker of gap junction integrity, within WT, sepsis induced a decrease in Vinculin levels, whereas no change was noted following sepsis within iNKT-/- mice.

Conclusion:
iNTK-cells affect the Angiopoietin pathway within the lungs, a pathway critical to maintaining the pulmonary vascular endothelial response to critical illness. Given currently available agents to both up- and down-regulate iNKT-cells, these findings offer a potentially novel therapeutic target for the amelioration of this morbid state.
 

58.02 TLR2 and TNF Induce Acute Kidney Injury Through Renal C3 Production in Diabetic Mice During Sepsis

Posted on January 18, 2018

L. M. Frydrych1, G. Bian1, K. He1, G. K. Wakam1, M. S. Anderson1, F. Fattahi2, P. A. Ward2, M. J. Delano1  1University Of Michigan,Department Of Surgery, Division Of Acute Care Surgery,Ann Arbor, MI, USA 2University Of Michigan,Department Of Pathology,Ann Arbor, MI, USA

Introduction:  Sepsis is the leading cause of death in the critically ill population. In diabetic patients with sepsis, acute kidney injury (AKI) frequently occurs and leads to increased mortality. Although the specific mechanisms responsible for diabetic AKI during sepsis are unknown, a growing body of evidence suggest that ongoing inflammation and renal complement deposition provoke chronic kidney injury in other disease states. However, the role that acute inflammation and complement deposition plays in the development of diabetic AKI following sepsis has yet to be investigated. We hypothesize that sepsis induced inflammation enables renal complement production, drives AKI evolution, and potentiates diabetic kidney injury following sepsis.

Methods:  30 week old C57BL/6, C3-/-, TLR2-/-, TNF-/-, and Diet Induced Obese (diabetic) mice with lean controls underwent sublethal cecal ligation and puncture (CLP) or sham procedure with and without TNF inhibition (Etanercept 100μg/mouse/day intraperitoneal). At serial time points between days 1 and 21 after CLP or sham, n=5 mice/group were euthanized. Kidneys and plasma were harvested for histology using in-situ hybridization or Periodic acid–Schiff staining, as well as analyzed for TLRs 1-9, C3, C5a, TNF, BUN, creatinine, and NGAL expression using RT-PCR or specific ELISA.

Results: In lean mice, AKI peaked 24 hours after CLP with a tenfold elevation in plasma and renal tissue NGAL levels compared to sham controls (p=0.001). In diabetic mice, AKI did not peak until 2 weeks after sepsis initiation. Renal C3, but not C5a, mRNA levels were eight fold higher 24 hours after CLP compared to sham in both lean and diabetic mice (p=0.001). However, in lean animals C3 levels returned to baseline at 7 days while C3 levels remained elevated, peaked at 2 weeks, and finally returned to baseline at 3 weeks in diabetic mice. Renal in-situ hybridization showed significantly more glomerular and proximal convoluted tubal C3 transcripts in septic diabetic animals compared to lean controls. RT-PCR analysis of kidney homogenates after sepsis revealed greater renal TNF mRNA expression over a 3 week period in diabetic compared with lean mice (p=0.01). TNF-/- and C3-/- lean mice each produced significantly less renal C3 and manifested less AKI, as determined by NGAL levels. TLR2 inhibition with TLR2-/- mice or Etanercept prevented renal C3 production (p=0.01), NGAL elevation (p=0.01), and sepsis induced AKI (p=0.01).

Conclusion: In lean mice, sepsis induced AKI occurs early, resolves quickly, and is dependent on local renal production of C3 through TLR2 and TNF signaling pathways. In diabetic mice, AKI also presents early; however, it persists for 2 weeks and does not fully resolve until week 3. TNF and TLR2 inhibition after sepsis represent novel therapeutic strategies to prevent kidney injury and improve diabetic survival following sepsis.

 

58.03 Systemic Regulation of Bone Marrow Stromal Cytokines Following Severe Trauma

Posted on January 18, 2018

E. S. Miller1, T. J. Loftus1, K. B. Kannan1, P. A. Efron1, A. M. Mohr1  1University Of Florida College Of Medicine,Department Of Surgery,Gainesville, FL, USA

Introduction: Severe traumatic injury induces a prolonged hypercatecholamine state which is associated with suppression of bone marrow erythroid progenitor colony growth that is mediated by the bone marrow stroma. This study sought to determine the effects of severe trauma on stromal hematopoietic cytokine expression. Utilizing human bone marrow stromal cells (HS-5 cells) cultured with plasma from healthy patients and trauma patients at a Level 1 trauma center, we hypothesized that trauma plasma would increase stromal expression of interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), and nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-kB).

Methods: HS-5 cells were cultured with the plasma of both healthy volunteers and trauma patients. At 6, 24, and 48 hours the expression of interleukin 6 (IL-6), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were measured using quantitative polymerase chain reaction. Statistical results were performed using GraphPad Prism. All data were presented as fold change mean±SD. Data significance was defined as *p<0.05 by t-test.

Results: IL-6 expression was increased at each time point with trauma plasma and IL-6 was significantly elevated when compared to normal plasma at 24 and 48 hours (Table). G-CSF and EPO expression were increased at all time points with trauma plasma and there was significant increase at 48 hours compared to normal plasma. Trauma plasma did not alter SCF expression at any time point. NF-κB expression increased from 6 hours to 24 and 48 hours with trauma plasma group. The addition of normal plasma and trauma plasma to HS-5 cells resulted in 89-100% cell viability.

Conclusion: Circulating plasma from trauma patients activated bone marrow stroma when compared to normal plasma. The addition of trauma plasma to human bone marrow stromal cells upregulated IL-6, G-CSF and EPO expression after 48 hours but did not affect SCF or NF-κB expression. This study demonstrates that there is systemic regulation of bone marrow stroma following severe trauma.

 

39.09 Better ABSITE Performance with Increased Operative Case Load During Surgical Residency

Posted on January 18, 2018

A. R. Marcadis1, T. Spencer1, D. Sleeman1, O. C. Velazquez1, J. I. Lew1  1University Of Miami,DeWitt Daughtry Family Department Of Surgery,Miami, FL, USA

Introduction: Common measures of evaluating surgical resident progression during General Surgery residency include American Board of Surgery In-Training Exam (ABSITE) scores and operative case logs. It remains unknown, however, if there is an association between operative case numbers and resident ABSITE performance. This study evaluates the relationship between operative cases performed and ABSITE scores at both the junior and chief resident levels. 

Methods: A retrospective review of ABSITE scores and operative case logs was performed for categorical General Surgery residents at a single institution at the junior (post graduate year [PGY]-2, n=45, from 2009-2017) and chief (PGY-5, n=19, from 2014-2016) levels. For each surgical resident, total number of operative cases logged (major and minor) from the start of their PGY-1 year until the end of either their 2nd or 5th year was calculated and compared to their ABSITE percentile score for that corresponding year using unpaired t-test and linear regression. Outliers with operative cases logged >3 standard deviations from the mean were excluded.

Results:  There was a linear relationship between total number of operative cases logged and ABSITE percentile score for surgical residents at both the junior (slope, m = 1.295) and chief resident (m = 6.109) levels, with a higher number of total operative cases logged being significantly associated with higher ABSITE percentile scores. For both junior and chief residents, there was a statistically significant difference in average number of operative cases logged between those with ABSITE scores below the 50th percentile and those with scores above the 50th percentile (junior cases, 311 vs. 370, p<0.05;  and chief cases, 1352 vs. 1683, p<0.05), respectively.

Conclusion: Surgical residents who perform higher numbers of operative cases do significantly better on the ABSITE than their peers with lower operative case numbers. This association may be due to increased clinical experience, exposure to pathology and/or individual surgical resident motivation.

 

39.10 Not All Operative Experiences Are Created Equal: 18 Year Analysis of a Single Center’s Case Logs

Posted on January 18, 2018

A. R. Cortez1, V. K. Dhar1, J. J. Sussman1, T. A. Pritts1, M. J. Edwards1, R. Quillin2  1University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA 2Columbia University College Of Physicians And Surgeons,Center For Liver Disease And Transplantation,New York, NY, USA

Introduction: As surgical education continues to evolve, so too does the need for surgical educators to better understand how residents learn. Previous analyses of national Accreditation Council for Graduate Medical Education (ACGME) operative log reports have shown that total operative volumes for graduating general surgery residents have remained stable over time, despite changes in duty hour restrictions. However, variability in subspecialty operative volume and its impact on resident training experience has not been well studied. 

Methods: ACGME operative log data from 1999 to 2016 for a single academic general surgery residency program were examined. All residents completed the Kolb Learning Style Inventory during their training and were subsequently classified as action-based or observation-based learners. Statistical analyses were performed using Wilcoxon rank-sum test, Chi-square test and linear regression analysis. A p-value <0.05 was considered to be statistically significant.

 

Results: Over the 18-year study period, 106 general surgery residents graduated from our training program. There were 92 action-based learners (87%) and 14 observation-based learners (13%). These two groups were similar in terms of race, sex and having a dedicated research experience (all p=NS). Linear regression analysis showed no change in total major cases during the 18-year study period (p=0.38). Subcategory analysis, however, revealed a significant increase in operative volume upon graduation in the following defined categories: skin, soft tissue and breast (+2.8 cases/year); alimentary tract (+10.1 cases/year); abdomen (+15.4 cases/year); endoscopy (+3.0 cases/year) and laparoscopy (+10.9 cases/year, all p<0.05). Conversely, a decrease was seen in the following defined categories: liver (-0.44 cases/year), vascular (-1.1 cases/year) and endocrine (-0.5 cases/year, all p<0.05). Learning style analysis revealed that action-based learners completed significantly more cases than observation-based learners in each of the domains in which operative volume increased (Figure).

Conclusions: While the total operative volume of graduates at our center has remained stable over the past 18 years, the operative experience of general surgery residents has become more narrowed toward a less subspecialized general surgery experience. These shifts may be disproportionally impacting trainees, as observation-based learners were found to operate less than action-based learners in select categories. Residency programs should therefore incorporate methods such as learning style assessment to identify residents at risk of having a suboptimal training experience.

39.08 Vascular Surgery trends among general surgery residents: ’09-‘16

Posted on January 18, 2018

A. T. Mierzwa1, S. F. Markowiak1, S. A. Toraby1, C. Das1, S. Pannell1, M. M. Nazzal1  1University Of Toledo Medical Center,Department Of Sugery,Toledo, OH, USA

Introduction:
The ACGME 80-hour work week, implemented in ‘03-’04, resulted in general surgery residents (GSR) having less exposure to vascular surgery cases in favor to other areas of training, particularly laparoscopic and alimentary tract cases. To rectify this, the ACGME defined category minimums for vascular surgery which increased from 44 to 50 cases per year with a focus on cases traditionally associated with general surgeons. The current trend in vascular care has been shifting from open procedures to endovascular with a decrease in general surgeons performing vascular procedures. With an expected increases in vascular cases nationwide and shortages of formally trained vascular surgeons, the need for general surgeons doing vascular work is unlikely to decrease. The aim of this study was to examine trends in vascular surgery exposure during general surgery residency to help assess the graduating general surgery resident’s preparedness.

Methods:
Data available from Accreditation Council for Graduate Medical Education (ACGME) reports for both General Surgery Residency (GSR), Integrated Vascular, Vascular Surgery Fellowship (VSF) from the years 2009-2016. Case trends were examined for any procedure that had an average number of cases per year greater than 1.5, in any year between ‘09 and ‘16. Trends among major categories in vascular surgery within the same time frame were additionally examined. Each GSR trend was plotted against Integrated and VSF to examine if the trends showed similar variations at each annual time point.

Results:
Carotid Endarterectomy cases are greater than 85% of total Cerebrovascular procedures annually. GSR’s exposure to these cases have decreased (13.6 to 9.8 cases/year), as opposed to increased trend observed in integrated and VSF case volume. Peripheral Obstructive and Vascular Access procedures showed similar trends (23.5 to 20 cases/year and 36.2 to 32.7 cases/year; respectively). Vascular Trauma procedures, however, have increased in both GSR (0.3 to 2.5 cases/year) and Integrated but decreased in VSF.

Conclusion:
Vascular surgery exposure has been limited due to the work-hour restriction with emphasis being placed on alimentary and laparoscopic procedures. Many basic vascular skills are a mainstay in non-vascular surgeries. Using these basic vascular skills, general surgeons can be confident in some of the minor procedures involved with trauma, transplantation, and safe surgical skills overall. With the expected increase in vascular surgery cases nationwide, some minor procedures will inevitably spill over to general surgeons. Our analysis indicates that a percentage (approx. 5-10%) of GSR will not meet the minimum requirement of cases logged for graduation.

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