86.19 A Pilot Study of Tamoxifen in Patients with Oesophageal Cancer

Posted on January 31, 2019

A. De Rosa2, W. Al-Khyatt1, C. Tufarelli3, R. Khan2, S. Iftikhar1  1Derby Hospitals NHS Foundation Trust,Derby, United Kingdom 2University of Nottingham,Faculty Of Medicine & Health Sciences,Nottingham, United Kingdom 3Leicester Cancer Research Centre, University of Leicester,Leicester Department Of Genetics And Genome Biology,Leicester, United Kingdom

Introduction:

Oesophageal cancer (OC) is a male dominant disease with a male: female ratio of 5:1 to 10:1. The gender bias is not attributable to differences in exposure to known risk factors alone. Recent in-vitro studies demonstrate oestrogen receptor expression, and the inhibitory effect of tamoxifen (a selective oestrogen receptor modulator) on OC cell growth. The aim of this pilot study is to determine the feasibility of a future randomised clinical trial by measuring the biological effect of short-term tamoxifen on OC growth in-vivo.

 

Methods:

In this single-arm, single-centre open study, patients with OC who were not candidates for surgical resection or further chemotherapy were included. Each patient received 80mg of tamoxifen for 4 days followed by 20mg thereafter until gastroscopy and biopsy of the tumour 4 weeks later. The biological response to treatment was assessed by immunohistochemistry using a monoclonal antibody to Ki67. The percentage change of Ki67 expression in pre-tamoxifen and paired post-tamoxifen biopsies was measured. The difference in percentage expression was analysed using the Student’s paired t-test. 

 

Results:

Over a 6-month period, 13 patients consented to the study; median (range) age = 79 (60 – 94). Five patients failed to complete the study and were excluded. Eight patients (6 male with adenocarcinoma and 2 female with squamous cell carcinoma) were included in the final analysis. No adverse events were recorded. After a median (range) duration of 30 (28 – 45) days of tamoxifen treatment, there was no change in the mean±SDKi67 expression between paired pre-tamoxifen (65±29.2%) and post-tamoxifen biopsies (65.6±31.9%, P > 0.05). Of the two women included, there was a decrease in the mean±SD Ki67 expression between pre-tamoxifen (51.5±12.0%) and post-tamoxifen biopsies (22.5±3.5%, P > 0.05).

 

Conclusion:

This pilot study demonstrates the methodology employed is both feasible and safe, and therefore could be used in the design of a randomised, multicentre phase II study to evaluate the clinical outcomes of tamoxifen in OC. Although the study was underpowered and failed to demonstrate a reduction in OC cell growth with tamoxifen treatment, a reduction in Ki67 expression was observed in biopsies of the two women included in the study. Thus any future phase II study design should also incorporate gender-specific subgroup analysis.

86.18 Enhanced Effect of Mitomycin C with HSP90 Inhibition and Hyperthermia in Colon Cancer Cell Lines

Posted on January 31, 2019

G. V. Georgakis1, N. Charisis1, F. Philanthope1, C. Preece1, M. Talamini1, J. Kim1, A. Sasson1, P. Carino-Thompson1  1Stony Brook University Medical Center,Surgical Oncology,Stony Brook, NY, USA

Introduction: Heated Intraperitoneal Chemotherapy (HIPEC) has been shown to improve outcomes in patients with several diffuse peritoneal cancer, including cancers of colorectal origin. The drugs that have been mainly used during this procedure are mitomycin C (MMC) and oxaliplatin. Since heat shock proteins (HSPs), especially HSP90, have been shown to be adundantly expressed by cancer cells, and their inhibition has been shown to induce apoptosis and cell cycle arrest in both in vitro and in vivo, we hypothesized that addition of the novel HPS90 inhibitor ganetespib, which is well tolerated and currently under clinical investigation, could increase the efficacy of chemotherapy in association with hyperthermia. 

Methods: We used two cell lines of colorectal origin (HCT116 and HT29) and we performed proliferation assays with the Cell Counting Kit-8 Cell Proliferation / Cytotoxicity Assay Kit with . Additionally, for molecular studies we perfomed western blots.

Results: HSP90 was fundamentally expressed by both HCT116 and HT29 cells. MMC and ganetespib as monotherapy were found to have an antiproliferative effect in a time and dose manner in HCT116 and HT29 cells. Additionally, hyperthermia  at 42C had a similar antiproliferative effect in a time manner. Combination of non lethal doses of MMC and ganetespib had a significant synergistic antiproliferative effect effect on both cell lines, which was more evident at 48 hours.

Conclusion: The HSP90 inhibitor ganetespib in hyperthermic conditions, potentiates the effect of chemotherapy (MMC), and may have a role in cytoreductive surgery and HIPEC.

 

86.16 The relationship between ANXA1 expression and breast cancer outcome is different in each subtype.

Posted on January 31, 2019

M. Okano1, E. Katsuta1, M. Oshi1, X. Peng2, L. Yan2, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Biostatistics & Bioinformatics,Buffalo, NY, USA

Introduction: Annexin A1 (ANXA1) is a phospholipid-linked protein, is known to have multiple functions related to inflammatory pathways, cell proliferation and the regulation of cell death signaling. ANXA1 was reported to have some association with cancer development, but the role of ANXA1 seems different depends on cancer types. Recently, we reported that ANXA1 is associated with triple-negative breast cancer (TNBC) and its poor prognosis in 211 Japanese breast cancer cases. Some reports similarly that ANXA1 high expressed breast cancer has poor survival. On the other hand, there are some studies demonstrating opposite results with high ANXA1 expression being associated with better outcome. It is speculated that the breast cancer subtypes may be one of the reasons for these controversial results. In this study, we investigated the association of ANXA1 mRNA/protein expression and patient survival in each subtype using gene and protein expression data of the publically available large cohort.

Methods: Clinical, RNA-seq and Reverse Phase Protein Array (RPPA) data were obtained from the Cancer Genome Atlas (TCGA). Overall survival (OS) and Gene set enrichment analysis (GSEA) were conducted comparing high and low expression group. 

Results:Among ER positive and HER2 positive patients, low mRNA expression of ANXA1 group has significantly worse OS (p=0.004, p=0.005, respectively). On the other hand, high mRNA expression of ANXA1 group showed significantly worse OS (p=0.028) in TNBC patients. In analysis using RPPA data, OS was significantly shorter in patients with high ANXA1 tumors among ER positive patients and HER2 positive patients (p<0.001, p=0.016, respectively) but high ANXA1 group has worse OS in TNBC patient (p=0.0095), which were in agreement with transcriptome analysis. To explore the mechanism of these results, GSEA was conducted. In TNBC patients, high ANXA1 expression tumors were enriched EMT related genes (NES=1.916, p=0.004), IL2/STAT5 (NES=2.04, p=0.003) and TNF-α signaling related genes (NES=2.02, p=0.011). On the other hand, in ER positive and HER2 positive patients, high ANXA1 expression tumors were enriched apoptosis related genes (NES=2.30, p<0.01) and p53 pathway related genes (NES=2.08, p<0.01).

Conclusion:We demonstrate high expression of ANXA1 enriched EMT signaling related gene expression in TNBC patients that associated with worse OS. On the other hand, in ER positive patients and HER2 positive patients, high ANXA1 expression is associated with better progression and the possible reason of this outcome is the apoptosis and p53 pathway. This discrepancy between ER positive breast cancer and TNBC on the effect of ANXA1 expression on patient survival might exemplify the fact that these subtypes possess significantly deferent molecular/genetic backgrounds.

 

86.15 IQGAP1/Rho-GTPase mechanotransduction pathway mediates adverse implant-tissue interactions

Posted on January 31, 2019

J. Padmanabhan1, T. Dohi1, Z. A. Stern-Buchbinder1, P. A. Than1, A. Trotsyuk1, S. H. Kwon1, Z. Maan1, G. C. Gurtner1  1Stanford University,Surgery,Palo Alto, CA, USA

Introduction.

Biomedical implants such as pacemakers, breast implants, and biosensors are employed to improve the quality of life for millions of patients worldwide. Implant performance and longevity is primarily limited by foreign body reaction (FBR), which is a fibrotic reaction characterized by accumulation of cells, proteins, and other biological materials at the implant-tissue interface. Severe FBR leads to formation of thick, collagen-rich FBR capsules, poor implant-tissue integration and implant rejection. We have previously shown that severe fibrosis during wound healing, a process closely related to FBR, is mediated by mechanical force (mechanotransduction). The role of mechanotransduction in FBR severity is not clear since currently used animal models do not recapitulate the mechanical environment around biomedical implants in humans. Moreover, which specific cellular subpopulations are involved and how mechanical cues activate these cells remain unknown. 

 

Methods.

We developed a novel surgical model to study mechanically-induced severe FBR in mice. Briefly, we manufactured cylindrical silicone implants which could be adapted to house small, prefabricated coin motors that can be powered using an external battery to induce implant vibration. Vibration-enabled implants and control silicone implants were implanted subcutaneously in mice for 2 and 4 weeks. Beginning on post-operative day 4, mice with vibration-enabled implants were sedated and their implants vibrated 1 hour daily for 8 days. FBR capsules from the murine models were compared to human FBR tissue using immunohistochemistry to validate the improved murine model.

Further, FBR tissue from the improved murine model were analyzed using single cell sequencing (scRNA-seq) and proteomics analyses to identify critical mechanotransduction pathways that mediate FBR. Finally, we collected and analyzed various biomedical implants (pacemakers, chest batteries etc.) explanted from human patients to confirm the findings.

 

Results.

The mechanical environment around biomedical implants in humans can be recreated in mice by using vibration-enabled silicone implants. This is an improved and easily reproducible model, which elicits human-like FBR to model silicone biomaterials in mice. We employed this improved model to study the role of mechanotransduction in adverse implant-tissue interactions.  scRNA-seq identified unique clusters of cells that highly express the IQGAP1/Rho-GTPase pathway, are upregulated in FBR. Comparative protein analyses of FBR capsules and control subcutaneous tissue confirmed the above findings. Additionally, IQGAP1/Rho-GTPase pathway components were detected at both transcript and protein levels in human FBR tissue,  further corroborating the animal data.

 

Conclusions.

IQGAP1/Rho-GTPase pathway is a promising target for therapies aimed at reducing FBR. Further studies are underway to confirm these findings and develop therapeutic strategies to limit FBR.

86.14 Sphingosine-1-Phospate Enhances Both Excisional and Burn Wound Healing with Less Scaring

Posted on January 31, 2019

M. AOKI2,3, T. Tsuge3, H. Aoki2,4, P. Mukhopadhyay2, H. Yamamoto5, N. M. Matsumoto3, E. Toyohara3, R. Ogawa3, K. Takabe1,2,6,7,8,9  1Roswell Park Cancer Institute,Division Of Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 2Virginia Commonwealth University,Division Of Surgical Oncology, Department Of Surgery,Richmond, VA, USA 3Nippon Medical School,Department Of Plastic, Reconstructive, And Aesthetic Surgery,Bunkyo, TOKYO, Japan 4Jikei University,Department Of Surgery,Minato, TOKYO, Japan 5Osaka University,Department Of Molecular Pathology,Osaka, OSAKA, Japan 6State University Of New York At Buffalo,Department Of Surgery,Buffalo, NY, USA 7Tokyo Medical University,Department Of Breast Surgery And Oncology,Shinjuku, Tokyo, Japan 8Yokohama City University,Department Of Surgery,Yokohama, KANAGAWA, Japan 9Niigata University Graduate School of Medical and Dental Sciences,Department Of Surgery,Niigata, NIIGATA, Japan

Introduction: Wound healing starts with the recruitment of inflammatory cells, which is followed by fibroblast activation and tissue construction. Deep dermal burn (DDB) can easily progress to a deeper injury due to infection through necrotic tissues and requires a long time to heal. A novel treatment that can enhance local immunity and promote epithelialization may achieve a breakthrough in burn management. S1P is a lipid mediator that promotes angiogenesis and cell proliferation, and is indispensable for local immunity. We investigated the roles of S1P in wound healing and its effects in DDB treatment. 

Methods:  A murine excisional wound splinting model was used. The mRNA expression of sphingosine kinase 1/2 (SphK1/2), and S1P receptor 1/2 (S1PR1/2) in the wound were measured. Rates of wound closure in SphK1-/- and S1PR2-/- were compared with wild type mice. The effects of nanoparticle-mediated topical SphK1 plasmid delivery were compared with control treatments. Rat DDBs were created by contact with 78°C  water for 10 seconds. The effects of topical application with control or S1P ointment were analyzed.

Results: SphK1 was highly expressed in murine wounds and SphK1-/- mice exhibited delayed wound closure along with reduced angiogenesis and inflammatory cell recruitment. Nanoparticle-mediated topical SphK1 overexpression accelerated wound closure, which was associated with increased angiogenesis, inflammatory cell recruitment, and various wound-related factors. SphK1 overexpression also led to less scarring, and suggested the interaction between tumor growth factor (TGF) -β1 and S1P. Topical application of S1P accelerated rat DDB wound closure with increased angiogenesis and macrophage recruitment.

Conclusion: SphK1 plays an important role in strengthening immunity, and promoting rapid wound healing with less scarring. Topical application of S1P promotes DDB wound healing by recruitment of macrophages and enhanced angiogenesis. S1P can be a candidate for a novel treatment for burn wounds to reduce necrotic tissues, prevent infection, and promote epithelialization.
 

86.12 Trends and Seasonality in Hospitalization Rates for Acute Surgical Pathologies in the U.S.

Posted on January 31, 2019

A. Shmelev1, S. C. Cunningham1  1Saint Agnes Healthcare,Department Of Surgery,Baltimore, MARYLAND, USA

Introduction:
Circannual variation in certain acute medical and surgical conditions is a well-known phenomenon and has been described across the world. Among the most commonly reported are acute intra-abdominal pathologies such as acute appendicitis, pancreatitis, cholecystitis, inflammatory bowel disease (IBD).  Various theories have been suggested to explain observed seasonal fluctuations in diseases rates. We aimed to describe seasonal variations in admission rates for a number of acute surgical diseases in the U.S. using a large administrative database.

Methods:
All admissions with primary diagnoses of acute appendicitis, diverticulitis, cholecystitis, pancreatitis, IBD, gastrointestinal ulcers (PUD) and bleeding (GIB), and bowel obstruction were pulled form AHRQ NIS (2000-2012; n=4.1 million). Monthly admission rates were calculated and seasonal trends decomposition was performed using LOESS or X-13 ARIMA procedures.

Results:
Acute intra-abdominal infections and IBD demonstrated the widest seasonal amplitude with peaks during summer months (the most prominent for acute infections) and troughs in cold season. GIB admission occurred predominantly in early spring with the fewest admissions in the end of summer. Nonbleeding PUD had plateaus during warm months and troughs in December. Regarding annual trends the fastest growth occurred for acute pancreatitis, diverticulitis, cholecystitis, IBD and bowel obstruction, followed by GIB. Conversely, admission rates for acute appendicitis and PUD have decreased over that time span.

Conclusion:
Analysis of large representative database re-demonstrated, that the incidence of most acute surgical conditions obeys certain circannual patterns. Literature indicated correlations of incidence trends of acute intra-abdominal pathologies with certain climatic parameters. Although no causation can be made based on these correlations, it is evident that diseases incidence is to a certain degree controlled by external factors which we can account for in resource planning. Additional, more granular analyses are necessary to uncover a possible role of air pollution, dehydration, circannual dietary changes and fluctuations in incidence of gastrointestinal infections, as a hypothetical cause of acute surgical pathologies.
 

86.11 Surgical Smoke Evacuators Do Not Eliminate Fire Risk from Alcohol-Based Skin Preparations

Posted on January 31, 2019

H. Carmichael1, J. Samuels1, K. Wikiel1,2, T. N. Robinson1,2, C. Barnett1,2, T. Jones1,2, E. L. Jones1,2  1University of Colorado,Department Of Surgery,Aurora, CO, USA 2Denver Veterans Affairs Medical Center,Department Of Surgery,Aurora, CO, USA

Introduction:  

Operating room fires are a “never event” that can cause life-threatening injuries. Oxygen, electrosurgical instruments (ignition source) and alcohol-based surgical skin preparations (fuel source), are present in nearly every surgical procedure and are the three necessary components of a fire.  Surgical suction and/or surgical smoke evacuators have the potential to reduce the concentration of alcohol vapors at the site of electrosurgical device activation, thus reducing fire risk.  Our aim was to compare the incidence of fires created by activating a monopolar instrument on ex vivo porcine skin prepped with alcohol-based surgical skin preparations, with and without smoke evacuation. 

Methods:  

A standardized, ex vivo model was created with a 15 x 15 cm section of clipped porcine skin. The monopolar electrosurgical device was activated at 30 Watts spray coagulation mode in 21% oxygen (room air) for 2 seconds immediately following skin preparation with two common alcohol based skin preparations, 70% isopropyl alcohol with 2% chlorhexidine gluconate (CHG-IPA) or 74% isopropyl alcohol with 0.7% iodine povacrylex (Iodine-IPA).  Results with no suction device were compared to standard wall suction 5 cm from the tip of the active instrument, as well as two standard monopolar devices with built-in smoke evacuators (SE-1 and SE-2). The presence of a fire was determined by the agreement of 2 of 3 observers and was confirmed with a thermal camera.

Results:
No fires were created using non alcohol-based skin preparations. With no suction, fires were generated in 60% (18 of 30) and 47% (14 of 30) of tests for both alcohol-based skin preparations.  Standard wall suction did not significantly reduce the incidence of fires with either preparation (43% vs. 60%, p=0.30 and 57% vs. 47%, p=0.61).  Use of both the SE-1 and SE-2 devices resulted in a reduction in the number of fires for CHG-IPA (p=0.001 and p=0.004) but not for Iodine-IPA.  Neither of the tested devices with built-in smoke evacuators eliminated the incidence of fires. 

Conclusion: 

Smoke evacuation devices reduce but do not eliminate the incidence of fires when used with chlorhexidine-alcohol surgical skin preparation. Smoke evacuation does not significantly reduce fires with Iodine-alcohol surgical skin preparation. Non alcohol-based preparations are the most effective way to prevent operating room fires.

 

86.10 The extent of scar formation is characterized by changes in keratinocyte proliferation

Posted on January 31, 2019

H. Li1, S. Balaji1, X. Wang1, M. Rae1, M. Chandramouli1, A. Blum1, M. Kodali1, P. Bollyky2, M. Butte3, S. Keswani1  1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Stanford University,Infectious Diseases And Microbiology And Immunology,Palo Alto, CA, USA 3University Of California – Los Angeles,Pediatrics,Los Angeles, CA, USA

Introduction: The normal course of wound healing in adults results in variably scar formation. However, there is an extreme variability among individuals in the degree of scarring to similar injury. The mechanisms that underlie this spectrum of scar phenotypes in healthy population are unknown. The objective of our study is to determine morphologic and functional phenotypic differences among healthy patients that produce variable scar phenotypes.

Methods: We collected abdominal skin tissue from female patients undergoing abdominoplasty to remove cesarean scars and compared them with normal skin from the surrounding area. Scars were scored by the Vancouver Scar Scale and classified into groups of low (score 1-3) and high (score from 6-9) scares. Tissue sections were analyzed using immunohistochemistry for cell proliferation marker Ki67 to determine functional differences in the normal skin and scar samples of low and high scar-forming patients.

Results:Our biobank is comprised of tissues from n=7 low scar, n=4 high scar forming patients. Preliminary analysis demonstrated that low-scar producing patients had 3.8 fold more proliferating keratinocytes in their normal skin than high-scar patients (12%+/- 2.6 vs. 3.17%+/-2.1 Ki67 positive cells to total epidermis cells; n=2 patients per group, 3 serial sections were analyzed). Similar differences were observed in the scar tissue of low- vs. high-scar patients’ scars (12.4% vs. 6.2%+/-1.4 Ki67 positive cells to total epidermis cells).

Conclusion:These data suggest that fundamental differences in keratinocyte proliferation among patients may be related to the patient propensity for scar formation. It implies keratinocyte proliferation plays important roles in wound closure (i.e. establishment of a barrier), regulates cytokine and ECM production in wound healing and scar maturation.

 

86.09 The extent of scar formation is characterized by changes in keratinocyte proliferation

Posted on January 31, 2019

H. Li1, S. Keswani1  1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Stanford University,Infectious Diseases And Microbiology And Immunology,Palo Alto, CA, USA 3University Of California – Los Angeles,Pediatrics,Los Angeles, CA, USA

Introduction:
The normal course of wound healing in adults results in variably scar formation. However, there is an extreme variability among individuals in the degree of scarring to similar injury. The mechanisms that underlie this spectrum of scar phenotypes in healthy population are unknown. The objective of our study is to determine morphologic and functional phenotypic differences among healthy patients that produce variable scar phenotypes.

Methods:
We collected abdominal skin tissue from female patients undergoing abdominoplasty to remove cesarean scars and compared them with normal skin from the surrounding area. Scars were scored by the Vancouver Scar Scale and classified into groups of low (score 1-3) and high (score from 6-9) scares. Tissue sections were analyzed using immunohistochemistry for cell proliferation marker Ki67 to determine functional differences in the normal skin and scar samples of low and high scar-forming patients.

Results:
Our biobank is comprised of tissues from n=7 low scar, n=4 high scar forming patients. Preliminary analysis demonstrated that low-scar producing patients had 3.8 fold more proliferating keratinocytes in their normal skin than high-scar patients (12%+/- 2.6 vs. 3.17%+/-2.1 Ki67 positive cells to total epidermis cells; n=2 patients per group, 3 serial sections were analyzed). Similar differences were observed in the scar tissue of low- vs. high-scar patients’ scars (12.4% vs. 6.2%+/-1.4 Ki67 positive cells to total epidermis cells).

Conclusion:
These data suggest that fundamental differences in keratinocyte proliferation among patients may be related to the patient propensity for scar formation. It implies keratinocyte proliferation plays important roles in wound closure (i.e. establishment of a barrier), regulates cytokine and ECM production in wound healing and scar maturation.
 

86.08 Histologic and Cellular Marker Characterization in Hidradenitis Suppurativa

Posted on January 31, 2019

J. L. Roberson1,2, S. Nisar1,3, B. C. Carney1,4, A. Alkhalil1, L. T. Moffatt1,4, J. W. Shupp1,3,4,5  1MedStar Health Research Institute,Firefighters’ Burn And Surgical Research Laboratory,Washington, DC, USA 2George Washington University School Of Medicine And Health Sciences,Department Of Surgery,Washington, DC, USA 3Washington Hospital Center,The Burn Center, Department Of Surgery,Washington, DC, USA 4Georgetown University Medical Center,Department Of Biochemistry And Molecular And Cellular Biology,Washington, DC, USA 5Georgetown University Medical Center,Department Of Surgery,Washington, DC, USA

Introduction:

Hidradenitis Suppurativa (HS) is a chronic inflammatory skin condition that presents as painful, draining abscesses which undergo repeated cycles of inflammation and healing with fibrosis. Hurley stage I is characterized by isolated nodules, stage II with recurrent inflammation and tract formation, and stage III involves coalesced tracts. At the present time, there is no definitive medical treatment for HS and a great deal of patients eventually undergo surgical resection. This study aims to evaluate the cytoarchitecture and cell surface markers present in HS patients to further understand the disease and identify potential therapeutic targets that may be used to stop HS progression, ultimately providing an alternative to surgery.

Methods:

Samples for cytoarchitecture analysis were formalin-fixed, paraffin embedded skin biopsies from 11 patients with HS who underwent surgical excision between May 2014 to June 2018. Six patients had stage III disease, one was stage I, and the rest were not staged. Histopathologic analysis included Hematoxylin and Eosin (H&E) staining and imaging at 10x magnification. These were compared with healthy human skin cytoarchitecture, analyzed as above, side-by-side.

Anti-CD3, a T-cell marker, and anti-CD31, a PECAM and vascular T-cell specific marker, were used for immunohistochemical (IHC) analysis. IHC was conducted in four HS and four healthy skin specimens with six regions of interest (three epidermal and three dermal) per sample.  Cellular quantification was performed at 40x magnification and compared with Student’s t-test.

Results:

Histopathologic examination showed that HS skin had a wider epidermal layer, extending into and engulfing the dermis, leaving intact dermal islands. Collagen fibers were disorganized compared with healthy skin. Other histological differences included formation of an epithelialized tract within the dermis and epithelialization around a hair follicle.  Neither apocrine, eccrine nor sebaceous gland histology was noted to be different between normal and HS skin.

Furthermore, there was extensive cellular infiltration and aggregation in the dermis of 10 out of 11 HS patients. IHC analysis revealed that, at the dermal level, HS lesions had a significantly greater quantity of CD3+ (324.29±139.28 vs. 14.93±16.32, p<0.0001), and CD31+ (322.15±155.46 vs. 2.84±5.56, p<0.0001) cells/mm2 than healthy skin samples.

Conclusion:

Epidermal and dermal cytoarchitecture of HS lesions differ in comparison to healthy skin. Furthermore, HS lesions demonstrate a significantly greater dermal lymphocytic infiltrate compared to healthy skin. Given that these samples are architecturally and cellularly consistent with HS, transcriptomic and immunohistochemical analysis for genes and proteins of interest are ongoing, with the goal of identifying potential non-surgical therapeutic targets.

 

86.07 Dysregulation of NOTCH signaling pathway in Hidradenitis Suppurativa in Comparison with Normal Skin

Posted on January 31, 2019

S. Nisar1,2, J. L. Roberson1,5, B. C. Carney1,3, A. Alkhalil1, L. T. Moffatt1,4, J. W. Shupp1,2,3,4  1Firefighters’ Burn and Surgical Research Laboratory, MedStar Health Research Institute,Washington, DC, USA 2The Burn Center, MedStar Washington Hospital Center,Washington DC, WASHINGTON DC, USA 3Georgetown University School of Medicine,Department Of Biochemistry,Washington DC, DISTRICT OF COLUMBIA, USA 4Georgetown University School of Medicine,Department Of Surgery,Washington DC, WASHINGTON DC, USA 5The George Washington University School of Medicine and Health Sciences,Department Of Surgery,Washington DC, WASHINGTON DC, USA

Introduction:
Hidradenitis Suppurativa (HS) is a chronic inflammatory skin disease that presents as recurrent abscesses causing fistulous tracts and scarring in areas of apocrine glands. This is painful and debilitating condition leads to poor quality of life. Etiology of HS is multifactorial. NOTCH signaling pathway, involved in embryological development, cell differentiation, and signaling, has been implicated in the pathogenesis of HS, but is poorly understood. This study evaluates dysregulation of NOTCH and related signaling pathways in HS in an effort to identify potential therapeutic targets.

Methods:
Skin biopsies of lesional HS were collected during surgical excision under an IRB-approved protocol. Normal skin was collected from otherwise to be discarded panniculectomy samples and used as control. RNA was extracted and quantified and cDNA was generated via RT-PCR from 3 normal and 4 HS skin biopsies. A PCR array, containing 84 genes involved in and related to NOTCH pathway, was used to identify genes that are dysregulated in HS. Candidate genes were identified if there was 2.5 fold change in gene expression, compared to normal skin, in at least 2 of the HS samples.

Results:
NOTCH array analysis identified 3 genes with 2.5 fold upregulation in 2 out of 4 samples. Function and average fold change of each is as follows: KRT1 maintains skin integrity, (avg=1.76), UbD is involved in proteosomal degradation (avg=0.98) and CCNE1 regulates cell cycle (avg=2.45).  Nine genes with 2.5 fold down-regulation were found. CCND1 regulates G1/S transition (avg=-3.23), Hey1 is involved in somite development (avg=-11.58), MFNG demarcates boundaries during embryological development (avg=-4.67), MMP7 is involved in tissue remodeling (avg=-5.10), Notch 4 leads to cell proliferation and differentiation (avg=-6.40), DLL4 encodes for NOTCH ligand (avg=-6.16), LFNG defines boundaries during embryological development (avg=-7.43), PPAR-g promotes adipocyte differentiation (avg=-13.92), and LMO2, has a role in yolk sac erythropoiesis (avg=-8.08). Furthermore, heat map anlaysis revealed one HS sample to have different gene expression profile compared to control and other HS samples (Figure).

Conclusion:
Genes involved in embryological development, and skin and adipocyte differentiation are dysregulated in HS samples when compared to normal skin. This is a novel finding and has not been previously reported. Furthermore, gene expression profile is different in one sample, likely due to an early stage of the disease. Work is ongoing to correlate the identified candidate genes with their respective protein levels by immunohistochemical analysis of formalin-preserved HS skin biopsies with the eventual goal of identifying molecular targets for treatment of HS.
 

86.06 Development of Malignancy-Risk Gene Signature Assay for Predicting Breast Cancer Risk

Posted on January 31, 2019

J. Sun1, W. Sun1, D. Chen2, J. Li2, R. Roetzheim1, C. Laronga1, M. C. Lee1  1Moffitt Cancer Center And Research Institute,Breast Oncology,Tampa, FL, USA 2Moffitt Cancer Center And Research Institute,Biostatistics And Bioinformatics,Tampa, FL, USA

Introduction:
The Gail Model estimates 5-year risk of developing breast cancer (BC) in unaffected women using health information. Our objective was to develop a clinical assay to quantify risk of BC occurrence in women receiving benign breast biopsy using a unique gene expression signature to estimate risk of subsequent BC.

Methods:
A 56-gene malignancy risk (MR) gene signature assay was validated for formalin-fixed paraffin-embedded (FFPE) tissue. Women who developed BC <2 years from a benign breast biopsy were identified and matched by age and biopsy date to unaffected controls with 2+ years of follow-up; biopsies were obtained at a single institution from 2007-2011. The MR was applied to benign breast and available BC specimens. Receiver operating characteristic curves were generated to determine diagnostic accuracy of the MR score and the Gail Model. BC risk was calculated by MR score only, Gail score only, and combined MR and Gail scores, then analyzed using leave-one-out-cross validation.

Results:

663 women with benign breast biopsies were identified. 100 women had concurrent BC biopsies and were excluded, leaving 563 women with benign results; 48 women developed BC (cases) within 2 years of biopsy. Follow-up data was collected at 2- and 5-year follow-up; 2 control patients developed BC between years 2-5 and were reclassified. 30 BC cases were matched to 60 unaffected controls, but analyzable tissue-based assays were only obtained for 17 benign “pre-cancer” case biopsies and 35 unaffected benign control biopsies due to poor cellularity of FFPE. 28 malignant tissue case assays were also obtained for comparison (Figure 1). Kruskal-Wallis rank sum test confirmed a difference between the three groups (Figure 1). MR assay signatures were higher in the case group than control group (p=0.09).

When comparing the MR and Gail scores, the MR score did not reach statistical significance when calculated for control and pre-case groups in two separate cohorts (n=52, p=0.246; n=43, p=0.75), while the Gail score suggested difference between the two groups (n=43, p=0.107, AUC=0.683). The MR score demonstrated poor predictive value when applied alone (AUC=0.538-0.613). However, the two tests demonstrated the best predictive value when combined (AUC=0.710).

Conclusion:
Due to small sample size, we were not able to demonstrate predictive capability of the MR signature alone; however, we demonstrated the feasibility of using FFPE gene expression assays to develop a predictive test for BC. Our current technique was limited by poor cellularity of benign breast samples. We intend to further investigate the combination of MR signature and Gail Model score as a more accurate risk assessment for women undergoing benign breast biopsy.

86.05 Exosomes as Mediators of the Fibrotic Response of Dermal Fibroblasts to Biomechanical Tension

Posted on January 31, 2019

H. V. Vangapandu1, N. Templeman1, D. Colchado1, A. Blum1, H. Li1, X. Wang1, E. Steen1, P. Bollyky2, S. Keswani1, M. Robertson1, C. Coarfa1, S. Balaji1  1Baylor College Of Medicine,Dept Of Surgery,Houston, TX, USA 2Stanford University,Palo Alto, CA, USA

Introduction: Wound responses involve fibroblast(FB)-mediated scar formation potentiated by mechanical tension. There is significant heterogeneity in how different people scar from similar injuries. Exosomes play an important role in cell communication which governs scarring. It is unknown if heterogeneous scarring is attributable to differences between FBs, how they respond to tension, and their intercellular communication. We hypothesize that there are differences in FB responses to tension that contribute to scar heterogeneity via exosomes.

Methods: Skin and paired scar tissue was obtained from women with C-section scars who underwent abdominoplasty. Scars were classified as "low" or "high" based on VSS (<3 vs. >6). Fetal skin was evaluated as non-scarring control. Sections were histologically stained (H&E; Trichrome). FBs were isolated from normal skin and scar, cultured on silicone membranes +/-10% static strain (24h), and evaluated for differences in proliferation(Ki67), fibrogenic potential(aSMA, fibrosis array) and genes implicated in exosome biogenesis(RAB27a-b;SMPD3). Exosomes from different scar phenotypes were analyzed(size-Zetasizer; NGS), and adoptively transferred into SCID murine skin wounds, and wound repair was evaluated. p-values by ANOVA; (n=3/group).

Results: High-scarring patient scars had thicker collagen bundles with interstitial FBs in dermis. These FBs also had a significantly higher proliferation rate(p<0.05) in vitro as compared to low and non-scarring FBs. PCR-array results showed that twice as many fibrotic genes were altered in scarFB vs skinFB in high-scar patients as compared to low-scar patients. Rab27a-b and SMPD3 expression was different in non-, low- vs. high-scarring patients skinFB. In low-scar patients, gene expression was similar between normal skinFB and scarFB, where as in the high-scar patients, Rab27a and SMPD3 levels were lower in the scarFB as compared to skinFB. Size distribution profile of exosomes was similar in skin and scarFB of low-scar patients. High-scar FBs produced exosomes larger in size and greater in abundance (p<0.05). NGS showed that there are baseline differences in small ncRNA of different FB-derived exosomes. While tension induced differential changes in a-SMA, Rab27a-b and SMPD and exosome size and cargo profiles in skinFB of different patients, notably, tension-induced expression of the normal skinFB was similar to its paired scarFB baseline phenotype. Lastly, non-scarring FB-derived exosomes resulted in regenerative wound phenotype in SCID dorsal wounds, whereas low and high-scar FB derived exosomes induced a corollary severity in scarring.

Conclusion: Our findings suggest that fibroblasts from different scarring phenotypes respond differentially to tension, including changes in exosome production that could influence fibrogenic phenotype and maintain the scar memory. Mining these exosome profiles will lead to novel scar therapeutics.

 

86.04 IL-6 may regulate adhesion formation induced by cecum cauterization in mice.

Posted on January 31, 2019

N. Uyama1, S. Wu1, M. Sudo1, E. Hatano1, H. Tsutsui1, J. Fujimoto1  1Hyogo College of Medicine,Surgery,Nishinomiya, HYOGO, Japan

Introduction: Postsurgical fibrous adhesions often result in severe complications of intestinal obstruction, female infertility, and chronic abdominal pain. We revealed that IFN-γ and PAI-1 played a pivotal role in adhesion formation by regulating the coagulation fibrinolysis system in mouse abdominal adhesion formation model (Nature Medicine 2008). In the present study, we investigated another molecular mechanism of adhesion formations by microarray analysis.

Methods: Cecum cauterization model was used in the present study. Intraabdominal adhesions were formed at 7 days after cecum cauterization by bipolar forceps. Immunohistochemistry for Ly6G (neutrophil) and collagen Type I (COL(I)) and triple immunostaining for podoplanin (PDPN), WT-1 (mesothelial cell markers) and αSMA (a myofibroblast marker) were performed to characterize cell type in adhesion areas. To determine the key molecules and signaling pathways for adhesion formations, microarray analysis was performed with damaged ceca at 0h, 3h, 12h, and 72h after operation. The kinetics of expression of IL-6, TNF-α, CXCL-2, TGF-β, COL(I) in damaged ceca was investigated by qRT-PCR. In in vitro experiments, effect of IL-6, TNF-α and TGF-β on expressions of TNF-α, CXCL-2, TGF-β and COL(I) by Met-5A cells (a human mesothelial cell line) and isolated human neutrophils was investigated by qRT-PCR. 

Results:Neutrophil accumulation in damaged ceca was found at 3h after operation. At day 7, COL(I) deposition, neutrophil accumulation and PDPN+WT-1+αSMA+ cells (activated mesothelial cells) were found in adhesion areas. As for microarray analysis, IL-6-, TNFα- and TGFβ-signalings were found to be elevated in adhesion formations. mRNA expression levels of IL-6, TNF-α, CXCL-2, TGF-β and COL(I) in damaged ceca were upregulated to about 450-fold, 18-fold, 18000-fold, 3-fold and 40-fold of control during 7days after surgery, respectively. In in vitro experiments, IL-6 significantly induced mRNA expression of TNF-α (2 fold) and CXCL2 (1.5 fold) in Met-5A cells. TNF-α significantly induced mRNA expression of TNF-α (8.2 fold) and TGF-β (5.9 fold) by neutrophils. TGF-β significantly upregulates mRNA expression of TGF-β (1.9 fold) and COL(I) (2.3 fold) by Met5A cells. Collectively, we assume that IL-6 may induce neutrophil accumulation through the production of CXCL2 by mesothelial cells. IL-6 may also stimulate TNFα production by mesothelial cells, which induce TGF-β production by neutrophils. In turn, TGF-β may stimulate production of TGF-β and COL(I) by mesothelial cells. 

Conclusion:Mesothelial cells and neutrophils may be key players of adhesion formations. On adhesiogenesis, IL-6 may induce neutrophil accumulation, eventually leading to collagen production presumably via activating cross-talk between neutrophils and mesothelial cells.

 

86.03 Inhibition of lncRNA GAS5 enhances diabetic wound healing

Posted on January 31, 2019

J. Xu1, J. Hu1, C. Zgheib1, M. M. Hodges1, K. W. Liechty1  1University Of Colorado Anschtuz Medical Campus,Pediatric Surgery,Aurora, CO, USA

Introduction: Impaired diabetic wound healing is associated with abnormal long non-coding RNA GAS5 (Growth Arrest-Specific 5) and chronic inflammation. We have previously shown that lncRNA GAS5 was up-regulated in diabetic wounds, and the persistence of the proinflammatory macrophage (M1) phenotype was mediated partly by GAS5/STAT1 pathway, indicating a potential role for GAS5 in the pathogenesis of diabetic wounds. We hypothesized that inhibition of GAS5 would promote the transition of macrophages from M1 to M2 phenotype and enhance the wound-healing process.

Methods:   Db/Db diabetic mice and Db/+ non-diabetic mice were wounded with an 8-mm punch biopsy and the wounds treated with a lentiviral vector containing either the green fluorescent protein (GFP) or sh-GAS5 transgene, an shRNA technology to knock down GAS5. Computerized planimetry was used to measure wound size daily. Loss function of GAS5 in RAW macrophage was achieved by transfection. Real-time PCR was applied to measure genes expression.

Results: Inhibition of GAS5 resulted in a significant increase in the rate of diabetic wound healing, (59% vs 99% in GFP-treated wounds at day 8 after injury; P < 0.002), and also improved the early phase of non-diabetic wound healing. GAS5 inhibition resulted in significantly decreased production of pro-inflammatory cytokines (M1 macrophage marker genes) including iNOS, IL1-beta, TNF-α and IL6 in RAW macrophage.

Conclusion: The relative level of lncRNA GAS5 in the wound plays a key role in the wound-healing response. Alterations in the wound level of GAS5 by inhibition, enhance healing by potentially transition of prolonged M1 macrophage to M2 macrophage in diabetic wounds, leading to decreased production of inflammatory cytokines and inflammation.

 

86.02 Different impacts of Bone Morphogenetic Protein expressions in Breast Cancer Patients’ Prognosis

Posted on January 31, 2019

K. Takabe1, E. Katsuta1, A. A. Maawy1, L. Yan2  1Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NY, USA

Introduction:  Bone morphogenetic proteins (BMPs) are members of the TGFβ family of signaling pathways and are known to be essential in fetal development, tissue differentiation and a multitude of cellular functions. In breast cancer, differential expressions are noted among different subtypes. BMPs are also known to regulate the epithelial to mesenchymal transition, tissue invasion and metastasis. Low expression of BMP7 is known to be associated with a more metastatic phenotype, especially bone metastases. However, the impact of comprehensive BMPs gene expressions on breast cancer patients’ survival is still understudied. This study aimed to investigate the association of BMPs gene expression with breast cancer patients’ survival using a ‘big data’ approach employing RNA sequencing from The Cancer Genome Atlas (TCGA).

Methods:  A total of 1093 treatment naïve breast cancers underwent genetic sequencing and the results of their sequencing stored in TCGA dataset. Overall survival (OS) was compared between high and low mRNA expression of indicated BMP related genes; BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP10, BMP15 and BMP receptors 1A (BMPR1A) and 1B (BMPR1B), based upon RNA-sequencing data of TCGA.

Results: TCGA cohort was representative of national breast cancer patients with respect to stage, pathology and survival. High expression of BMP1 (p<0.001), BMP3 (p=0.002), BMP5 (p=0.020), BMP7 (p<0.001) and BMPR1A (p<0.001) significantly associated with better OS. On the other hand, high expression of BMP6 (p<0.001) and BMPR1B (p=0.005) significantly associated with worse OS. BMP2, BMP4, BMP10 and BMP15 did not associate with OS. When we analyzed by subtype, in consistent with the whole cohort analysis result, BMP7 high expression significantly associated with better prognosis in both ER positive (p<0.001) and negative tumors (p<0.001). Interestingly high expression of BMP6 associated with better prognosis in ER positive tumor (p=0.004), whereas it associated with worse prognosis in ER negative tumors (p=0.006).   

Conclusion: BMP expression profiles may be of value in prognostication. Intervention in this pathway may serve to improve outcomes, manage metastatic disease and assist in clinical decision making on optimal therapy based on risk of recurrence or metastasis.

 

86.01 Regulating Hyaluronan Deposition Attenuates Tubulointerstitial Fibrosis in Ureteral Obstruction

Posted on January 31, 2019

X. Wang1, S. Balaji1, H. Li1, E. Steen1, P. Bollyky3, J. Cheng2, S. Keswani1  1Baylor College Of Medicine,Pediatric Surgery/Surgery,Houston, TX, USA 2Baylor College Of Medicine,Nephrology/Medicine,Houston, TX, USA 3Stanford University,Infectious Diseases/Medicine,Palo Alto, CA, USA

Introduction: Renal fibrosis is a pathological characteristic of chronic kidney disease (CKD), and is a product of aberrant inflammation, extracellular matrix (ECM) deposition and peritubular capillary loss. We have demonstrated a novel role for interleukin-10 (IL-10) in abrogating dermal fibrosis by regulating hyaluronan (HA) through dermal fibroblasts. We therefore hypothesized that hyaluronan attenuate renal fibrosis via its molecular weight variation to influence extracellular matrix remodeling, promoting angiogenesis and reducing inflammation. 

Methods: In vivo: We performed unilateral ureteral obstruction(UUO) as a renal fibrosis model with/without IL-10 overexpression through the injection under the kidney capsule with normal diet or diets with 5% HA inhibitor, 4-methylumbelliferone(4MU). UUO/sham kidneys were collected at d3, d7, d21 for RNA, ELISA, and/or immunohistochemistry. HA synthesis and degradation enzyme levels were assessed by qPCR and Western blot. HA molecular weight was assessed by size-exclusion chromatography. In vitro: Renal fibroblasts (FB) were isolated from C57BL/6J mice to determine the effect of IL-10(100 ng/ml) on gene expression of HAS1-3 and hyaluronidases (HYAL1-2) at 24h.  Alpha-SMA, HAS1, HAS2 and p-STAT3 expression were assessed by western blotting at 48h. Data mean+/-SD; p-values by ANOVA. 

Results:
In vivo, the up-regulation of HAS1 and HAS2 expression in normal and 4-MU diets UUO mice compared to control mice from day 3; ELISA showed that total HA is steady increased from day 3 up to day 14 for UUO mice, and up to day 21 for IL-10 treated UUO mice. In normal diet mice, lenti-IL-10 treatment resulted in less dilated tubules, decreased kidney fibrosis, and preserved tubular integrity in kidneys, compared to control treated mice; IL-10 treated 4-MU diet mice were not able to achieve attenuated fibrosis.). HA gel electrophoresis showed Day 3, 7 and 14 UUO kidneys have a previously unreported 1.5×106kD HA variant compared to control/sham kidneys. In vitro: HAS1, 2&3 and HYAL1 in renal FB (6.62±0.89, 1.83±0.54, 1.84±0.92) was also significantly dysregulated (p<0.05) after 24h IL-10 treatment by qPCR.With western blotting, HAS2, a-SMA and STAT3 were significantly upregulated. A 1.88-fold increase in HA-rich matrix formation was shown with 24h IL-10 stimulation, and the effect was abrogated by HYAL (p<0.05). 

Conclusion:

Our study provides the first evidence that injured kidney tissues have the capacity to express increased levels of a high molecular weight HA variant, which contrasts to that found in normal, uninjured kidneys. This finding suggesting that HA plays important roles in kidney, development, homeostasis, architectural integrity and function.Moreover, our discovery of mechanisms behind the HA-attenuated fibrosis could inspire novel therapeutics.

 

67.10 Rapid Detection of Clostridium difficile Toxins in Stool by Raman Spectroscopy

Posted on January 31, 2019

S. Koya1, M. Brusatori1, S. Yurgelevic1, C. Huang1, L. N. Diebel2, G. W. Auner2  1Wayne State University,Smart Sensors And Integrated Microsystems, Michael And Marian Ilitch Department Of Surgery,Detroit, MI, USA 2Wayne State University,Michael And Marian Ilitch Department Of Surgery,Detroit, MI, USA

Introduction:
Clinical practice guidelines define Clostridium difficile infections (CDI) as diarrhea (≥3 unformed stools in 24 hrs.) with either a positive C. difficile stool test or detection of pseudomembranous colitis. Pathogenicity of CDI is due to toxins, toxin A (TcdA) and toxin B (TcdB). While presence of toxin is necessary for disease, detection of toxins using immunoassays is complex and lacks sensitivity. For this reason, positive C. difficile in stool by toxigenic culture (TC) and nucleic acid amplification testing (NAAT) are used. These tests are confounded by the presence of asymptomatic colonization of toxigenic C. difficile and leads to overdiagnosis of CDI. Raman spectroscopy (RS) is a novel technology that is used to detect bacteria and their toxins. RS doesn’t require antibodies for detection of toxins. We hypothesis that RS may be a sensitive method to detect C. difficile toxins in stool and solve overdiagnosis of CDI.

Methods:
CDI negative stool samples were spiked with varying concentrations of TcdA and TcdB. RS was performed on smear of stool on a mirror polished stainless-steel slide. RS of feces is difficult due to confounding background material and autofluorescence. The samples were photo-bleached to reduce autofluorescence. Raman spectra were obtained, background corrected, vector normalized and analyzed by machine learning methods including Support Vector Machine (SVM), Random Forest (RF) and Partial Least Square Linear Discriminant Analysis (PLS-LDA). The best model was chosen, and its accuracy was measured by train-test, cross-validation and bootstrap methods.

Results:
At 1ng/ml concentration, TcdA and TcdB spiked stool were distinguished from control stool by all models with various accuracies. SVM with linear kernel performed best for TcdA with an accuracy of 85% and and PLS-LDA performed best for TcdB with 75% accuracy.

Conclusion:
RS of feces is difficult due to autofluorescence. Despite the difficulty, RS successfully detected TcdA and TcdB in stool samples. The autofluorescence could be further decreased by using diluted samples of stool and the accuracy of the separation could be increased by deep learning algorithms. Thus, RS has the potential to rapidly detect C. difficile toxins in stool at clinically relevant concentrations, be a point of care diagnostic tool and solve overdiagnosis of CDI.  
 

67.09 UCH-L1 IS A SERUM BIOMARKER FOR PERIPHERAL NERVE INJURY IN A MODEL OF EXTREMITY ISCHEMIA

Posted on January 31, 2019

A. P. Bercz1, M. C. Morris1, F. Kassam1, R. Veile1, L. Friend1, R. Schuster1, T. A. Pritts1, A. T. Makley1, M. D. Goodman1  1University Of Cincinnati,Department Of Surgery, College Of Medicine,Cincinnati, OH, USA

Introduction:  Ubiquitin carboxy-terminal hydroxylase L1 (UCH-L1) is specifically expressed by neurons and its release reflects the neuronal response to injury. In humans and murine models, UCH-L1 is elevated in serum and cerebrospinal fluid following traumatic brain injury (TBI), hypoxic encephalopathy, and intracerebral hemorrhage, but it is unknown whether UCH-L1 is specific to central nervous system injury. Our lab has recently demonstrated that UCH-L1 is significantly elevated in a murine polytrauma model, regardless of TBI. In this study, we hypothesized that UCH-L1 would function as a marker of peripheral nerve injury induced by extremity ischemia.

Methods:  Mice were anesthetized mice with pentobarbital and the left femoral vessels isolated. Mice were then subjected to either femoral artery isolation alone (sham), femoral artery ligation, femoral vein ligation, femoral artery ligation after 60 minutes of controlled hemorrhagic shock followed by resuscitation with shed blood, or four hours of ischemia with a femoral artery clamp followed by reperfusion (I/R). Mice were sacrificed at 4, 24, or 72 hours after treatment. Serum was analyzed using enzyme-linked immunosorbent assays for UCH-L1, neuron specific enolase (NSE), and creatine kinase (CK), the latter two being known biomarkers of neuronal and muscle injury, respectively. The bilateral quadriceps muscles were harvested and analyzed using hematoxylin and eosin staining.

Results: UCH-L1 was significantly increased in the hemorrhagic shock/resuscitation group at 4 hours, compared to the sham, artery ligation, vein ligation, and I/R groups (Figure 1). No other groups had significant UCH-L1 elevations compared to Sham at 4 hours. In the setting of hemorrhagic shock, UCH-L1 elevation persisted through 72 hours. NSE was not as sensitive to these ischemic changes. CK was altered in a pattern similar to that of UCH-L1 in its temporal response profile and was significantly increased in the hemorrhagic shock/resuscitation group with respect to the other groups at 4 hours. Hematoxylin and eosin staining of the quadriceps revealed muscle necrosis in the hemorrhagic shock/resuscitation group only, demonstrated by increased inflammatory infiltrate surrounding enucleated fascicles.

Conclusion: The acute elevation of UCH-L1 in response to isolated, controlled hemorrhagic shock and resuscitation suggests that UCH-L1 is not specific for TBI or central nervous system injury. The lack of NSE elevation in response to femoral vessel ligation suggests that NSE may be a more specific biomarker for TBI than peripheral nerve injury. UCH-L1 may be utilized as an early marker of neuronal injury in the context of hemorrhagic shock, polytrauma, and isolated extremity ischemia.

 

67.08 The Role of Chemoprophylactic Agents in Modulating Hypercoagulability after Traumatic Brain Injury

Posted on January 31, 2019

F. Kassam1, M. C. Morris1, A. Bercz1, R. Veile1, L. Friend1, N. Beckmann1, C. C. Caldwell1, M. D. Goodman1  1University Of Cincinnati,Department Of Surgery, College Of Medicine,Cincinnati, OH, USA

Introduction:  The pathophysiology behind the subacute but persistent hypercoagulable state following traumatic brain injury is poorly understood but contributes to morbidity induced by venous thromboembolism (VTE). Because platelets and their microvesicles have been hypothesized to play a role in posttraumatic hypercoagulability, administration of commonly utilized agents for inflammation, VTE chemoprophylaxis, and sphingolipid modulation may ameliorate this coagulability. We hypothesized that utilization of aspirin, ketorolac, amitriptyline, unfractionated heparin, and enoxaparin would modulate the platelet and whole blood coagulation response following traumatic brain injury.

Methods:  A standard weight-drop system was utilized to induce concussive traumatic brain injury in mice. Following injury, mice were randomized into drug treatment groups to receive aspirin (100 mg/kg), ketorolac (5 mg/kg), amitriptyline (10 mg/kg), heparin (75 IU/kg), enoxaparin (3 mg/kg), or saline control (100µL) at 2 and 8 hours post-TBI. Mice were then sacrificed at 6 or 24 hours after injury and blood was drawn via cardiac puncture to determine coagulability by thromboelastometry (EXTEM and FIBTEM), platelet function testing with impedance aggregometry, and microvesiscle enumeration utilizing nanoparticle tracking analysis.

Results: Thromboelastometry results demonstrated that the platelet contribution to maximum clot firmness (%MCF-Platelet) at 6 hours (Figure 1) was significantly higher in mice that received aspirin (69%, p<0.002) or amitriptyline (68%, p<0.007) compared to mice that received saline (57%). At 24 hours, the %MCF-Platelet remained significantly higher in mice that received amitriptyline (66%, p=0.04) compared to those that received saline (63%). The overall ADP- and arachidonic acid-induced platelet aggregation was significantly lower in mice receiving ketorolac,  aspirin, and amitriptyline compared to mice receiving saline at 6 hours post-injury. By  24 hours after injury, mice that received aspirin (40 a.u., p<0.005), ketorolac (38 a.u., p=0.02), and enoxaparin (35 a.u., p=0.04) had significantly lower ADP-induced platelet aggregation than saline control mice (54 a.u.). However, there was no difference in the total or percentage of platelet-derived (CD41+) microvesicles between any treatment group at both 6 and 24 hours.

Conclusion: Following traumatic brain injury, amitriptyline decreased platelet aggregability and increased contribution to clot in a manner similar to aspirin. The effect of amitriptyline on platelet function reflects a possible role of acid sphingomyelinase in the hypercoagulability observed following injury and suggests sphingolipid metabolism as a novel target for multimodal VTE chemoprophylaxis. Additionally, inhibition of platelet reactivity may be an underappreciated benefit of low molecular weight heparins, such as enoxaparin, compared to unfractionated heparin use.

 

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