58.05 The Creation and Implantation of Three-Dimensionally (3D)-Printed Vascular Patch Grafts

Posted on January 26, 2017

B. C. Wengerter1, G. Emre1, P. J. Geibel1, H. Bai1, A. Dardik1, J. P. Geibel1  1Yale University School Of Medicine,Surgery,New Haven, CT, USA

Introduction:

Cardiovascular disease carries a significant morbidity and mortality burden in the United States. Surgical options for vascular grafts have traditionally included autologous vein or synthetic graft material. Three-dimensional (3D) bioprinting provides a promising alternative when the aforementioned materials fail or are unavailable.

Methods:

Rat fibroblast and smooth muscle cells were encapsulated in a gelatin and hyaluronic acid hydrogel to create bioink, which was then used to print a 280 mm2 cellular sheet via a novel three-dimensional printing technique. Printed structures were allowed to mature for a period of seven days, after which they were fashioned into vascular angioplasty patches for repair of inferior vena cava venotomy in male Wister rats. The rats were sacrificed seven days post-implantation, and the implants with surrounding inferior vena cava were excised and analyzed by histology and immunohistochemical staining.

Results:

The rats thrived for seven days post-implantation, with normal fluid intake and weight gain. The patched inferior venae cavae showed no gross evidence of hemorrhage or thrombosis at time of excision. Patches were well incorporated into the host vessels and demonstrated an organizing structure consistent with their early post-implantation state. Neointima formation was evident by von Willebrand factor staining, and staining for lymphocytes and macrophages demonstrated only a mild inflammatory reaction.

Conclusion:A 3D-printed biologic graft survives early implantation for inferior vena cava patch angioplasty without any evidence of thrombosis, hemorrhage or immediate immunologic rejection. It may provide a viable alternative both for patching and for larger-scale reconstruction.

58.04 Physiological Levels of Resistin Stimulate Inflammation in Macrophage and VSMC Co-culture via PKC-ε

Posted on January 26, 2017

M. C. Zuniga1,2, G. Raghuraman1,2, W. Zhou1,2  1VA Palo Alto Healthcare Systems,Vascular Surgery,Palo Alto, CA, USA 2Stanford University,Vascular Surgery,Palo Alto, CA, USA

Introduction:  Resistin, an adipokine with inflammatory properties, has been associated with atherosclerosis and cardiovascular disease. However, its specific mechanisms of action in key cells involved in atherosclerotic plaque formation are not well defined. We have shown that resistin promotes vascular smooth muscle cell (VSMC) dysfunction at a pathological concentration. The purpose of this study was to examine the direct effect of resistin on macrophage-related inflammation and its indirect effects on VSMCs in the presence of macrophages.

Methods:  Human monocytes were isolated from healthy controls and differentiated into macrophages (MΦ). MΦ were treated with resistin at a “high” physiological level (10 ng/ml) for 18 h, with or without selective PKC-ε inhibitor, εV1-2, at 1 µM. MΦ supernatants (conditioned media) were analyzed with ELISA for pro-inflammatory cytokines. MΦ were assessed for gene and protein expression of inflammatory markers using RT-PCR, immunocytochemistry (ICC) and Western blot, respectively. Conditioned media (CM) generated from MΦ cultures were used to treat human coronary artery smooth muscle cells (HCASMCs) for 24 h.  HCASMCs were evaluated for changes in proliferation using the MTT assay and flow cytometry, as well as gene expression changes of nuclear factor-kappa-B (NF-kB) subunits and of proliferation-associated genes.

Results: Physiological levels of resistin triggered increased gene expression and protein production of the inflammatory cytokines TNF-α, CD40L, IL-6, and resistin in MΦ (Figure 1 A-D). MΦ showed significantly upregulated expression of membrane receptor CD40 and downregulated CD206 (mannose receptor) after resistin treatment. Inhibition of PKC-ε reduced gene expression of inflammatory cytokines and protein expression of CD40. HCASMCs exposed to CM from resistin-treated MΦ showed significantly increased proliferation, while blocking PKC-ε in MΦ during resistin treatment reduced CM-induced proliferation in HCASMCs (Figure 1 E). Furthermore, HCASMCs upregulated gene expression of NF-kB1 (p50), NF-kB2 (p52), cyclin-D1 and proliferating cell nuclear antigen, whereas this effect was not observed in HCASMCs treated with CM from control MΦ.

Conclusion: Resistin at a high physiologic concentration promotes pro-inflammatory MΦ transformation via PKC-ε, and it induces VSMC dysfunction indirectly through MΦ. Targeting resistin may represent a therapeutic strategy in atherosclerosis-associated complications. Further in vivo investigations are warranted.

 

58.03 A Novel Model of Aortic Aneurysm Rupture

Posted on January 26, 2017

A. Z. Fashandi1, M. D. Salmon1, R. B. Hawkins1, M. Spinosa1, G. Lu1, G. Su1, G. Ailawadi1, G. R. Upchurch1  1University Of Virginia,Department Of Surgery,Charlottesville, VA, USA

Introduction:  Given the relative unknown biologic antecedents that occur prior to aortic aneurysm rupture in humans, the purpose of this study was to establish a reproducible murine model of aortic aneurysm (AA) rupture.

Methods:  Seven-week-old apolipoprotein E deficient (ApoE-/-) mice were fed a high fat diet for 4 weeks and subcutaneous osmotic infusion pumps containing Angiotensin II (ATII) were then implanted. ATII was delivered continuously for 4 weeks at either 1000ng/kg/min (n=25) or 2000ng/kg/min (n=29). A third group of mice (n=14) was given ATII at 2000ng/kg/min and 0.2% β-aminopropionitrile (BAPN) dissolved in the drinking water. Surviving mice were euthanized 28 days after osmotic infusion pump placement and aortic diameters were measured. All animals that died prior to 28 days underwent autopsy to determine cause of death. Survival was analyzed by Kaplan-Meier while rates of aortic rupture and aneurysm formation were analyzed with Chi-squared or Fisher’s exact test, where appropriate. Statistical analysis was performed with GraphPad Prism 7.0 and significance was set at a of 0.05.  

Results: Survival was significantly different among the three groups with 80% survival at 28 days in the 1000ng/kg/min group, 52% in the 2000ng/kg/min group, and only 14% survival in the ATII/ BAPN group (p=0.0001; Figure 1). Unadjusted comparisons between each group were also significantly different (p<0.05). Rupture rates were statistically different among groups (8% vs 38% vs 79%, p<0.0001) and were again different on unadjusted comparison between groups (p<0.03). Incidence of abdominal AA formation were 48%, 55% and 93% in the three experimental groups and there were statistically significant differences between the ATII/ BAPN group and both the 1000ng/kg/min ATII and the 2000ng/kg/min ATII groups (p=0.006 and p=0.0165, respectively). Finally, rates of ascending thoracic AA formation were 12%, 52% and 79% in the three experimental groups with statistically significant differences between the 1000ng/kg/min ATII group and both the 2000ng/kg/min ATII and the ATII/ BAPN groups (p=0.0033 and p<0.0001, respectively). 

Conclusion: In this study, a reproducible model of aortic rupture was developed with a high incidence of both abdominal and thoracic aortic aneurysm formation. This model should enable further studies to expand on research in aortic aneurysms and the pathogenesis of aortic rupture, as well as targeted strategies to prevent human aortic aneurysm rupture. 

 

58.02 Perivascular Delivery of Resolvin D1 Inhibits Neointimal Hyperplasia in a Rabbit Vein Graft Model

Posted on January 26, 2017

B. Wu1, A. Chatterjee1, G. Mottola1, E. Werlin1, M. Chen1, K. D. Lance2, T. A. Desai2, M. S. Conte1  1University Of California – San Francisco,Vascular Surgery,San Francisco, CA, USA 2University Of California – San Francisco,Bioengineering,San Francisco, CA, USA

Introduction:  Persistent inflammation following vascular injury is a driver of neointimal hyperplasia. Recent work demonstrates that specialized lipid mediators derived from omega-3 polyunsaturated fatty acids, such as Resolvin D1 (RvD1), actively orchestrate the process of resolution. We investigated local perivascular delivery of RvD1 in a rabbit vein graft model.

Methods:  Ipsilateral jugular veins were implanted as carotid interposition grafts via an anastomotic cuff model in New Zealand white rabbits (3-4 kg; N=68). RvD1 (1000 ng) was delivered to the vein bypass grafts in a perivascular fashion, using either 25% Pluronic F127 gel or a thin bi-layered biodegradable poly-lactic-co-glycolic acid (PLGA) wrap. Drug delivery to rabbit veins was confirmed ex-vivo by LC-MS-MS and ELISA. Leukocyte infiltration and cell proliferation were evaluated at 3 days post-bypass via immunohistochemistry. Morphometric analysis was conducted at 28 days post-bypass to evaluate vein graft remodeling. 

Results:  Perivascular constructs did not influence rates of graft thrombosis, wound infection or death. Leukocyte (CD45) and macrophage (CD68) infiltration was significantly reduced in the RvD1-gel group versus bypass-only and vehicle-gel groups at 3 days (60-72% reduction, P < .01). Cellular proliferation (Ki-67 index) was significantly lower in RvD1-gel grafts versus both bypass-only and vehicle-gel grafts at 3 days (43% and 45% reduction respectively, P < .01). Treatment of vein grafts with RvD1-loaded gels reduced neointimal thickness at 28 days by 61% versus bypass-only grafts (P < .001) and by 63% versus vehicle-gel grafts (P < .001). RvD1-loaded PLGA wraps reduced neointimal formation at 28 days by 50% versus the bypass-only group (P < .001). There was a trend towards decreased graft neointimal thickness for RvD1-loaded wraps versus vehicle-wraps at 28 days, however this did not reach statistical significance (32% reduction, P = .085) [Figure]. 

Conclusion:  Local perivascular delivery of the pro-resolving lipid mediator RvD1 attenuates formation of neointimal hyperplasia without associated toxicity in a rabbit vein graft model. This effect appears to be mediated by both reduced leukocyte recruitment and attenuated cell proliferation in the graft. Perivascular PLGA wraps may also impart protection via external biomechanical scaffolding in this model. Our studies provide further support for the potential therapeutic role of D-series resolvins in modulating vascular injury and repair.

42.20 Development of Hepatic Injury Model in Rats for Testing Hemostatic Patches

Posted on January 25, 2017

E. Roozen1, R. Lomme1, H. Van Goor1  1RadboudUMC,General Surgery,Nijmegen, GELDERLAND, Netherlands

Introduction:
Intra-operative or traumatic bleeding are common problems in surgery causing significant morbidity and mortality. Hemostatic products are increasingly recognized as an important measure to control bleeding. There is a need for safe, synthetic, cheap and effective alternatives for the already available hemostatic products. Preclinical testing of these products is predominantly performed in pig liver injury models. We develop hepatic injury models in small animals in order to replace the pig model. These models are our first step to ultimately create ex vivo perfusion models in order to reduce, refine and replace (3R’s) animals.  Aim of this study is to explore the consistency in creating a significant bleeding defect of the rat liver to discriminate between different hemostatic patches in terms of efficacy.

Methods:
Two models were evaluated, a liver biopsy punch model and a partial liver resection model . It is known that punch biopsies are reproducible because the surface area and depth have the same dimensions and can be controlled for. Resection models vary more, but have more clinical relevance. Thirty rats were used, in each rat 2 defects were created. In experiment 1, different punch diameters and depths were used to obtain a reproducible and significant bleeding (punch model). In another set of animals partial-lobe resection was performed (resection model). Rats were randomized for either model, receiving an active patch (A1) or a control non-active patch (P1) on both defects. In experiment 2 the feasibility of folding the patch around the edges of the resected area was explored using 2 active patches (A2 and A3).
Outcome for consistency were the severity of bleeding, amount of bloodloss (BL,weight absorbed blood), the surface area of the defect (photodigital planimetry), and the weight of the resected specimen. Outcome for efficacy were time to hemostasis (TTH) and BL after patch application, prior to hemostasis.

Results:
A punch diameter of 8x3mm and the resection created consistent bleedings. 37/60 defects were evaluable for efficacy. There was no difference in TTH (p=0,715) and BL (p=0,440) between the A1 and P1 in the punch model, whereas A1 significantly decreased TTH (p=0,003) and BL (p=0,001) compared to P1 in the resection model. Non-folding was better feasible than folding the patches without differences in TTH and BL. A2 and A3 patches differed in TTH (p:0,02) and BL in both the folding (TTH: p=0,02; BL: p=0.049) and non-folding (TTH: p=0,000 ; BL: p=0.005) application.

Conclusion:
In rats a consistent and easy to operate, partial liver resection model can be created that discriminates between well and worse performing hemostatic products using easy to measure and clinically relevant outcomes.
 

42.19 Ciclopirox Olamine Causes Growth Inhibition of Esophageal Carcinoma Cells

Posted on January 25, 2017

R. J. Ryan1, P. Ramamoorthy1, S. J. Weir2, S. Anant1, P. DiPasco1  1University Of Kansas,General Surgery,Kansas City, KS, USA 2University Of Kansas,Pharmacology, Toxicology And Therapeutics,Kansas City, KS, USA

Introduction:  

Esophageal carcinoma is a devastating diagnosis with an overwhelmingly poor prognosis. More than 30% of patients have metastatic disease on presentation, and 5 year survival in these patients is 3.5%. Chemo-radiotherapy is the primary treatment modality of advanced disease, but current chemotherapeutic agents only prolong life for months to a few years. Drug repositioning is an important pharmacologic strategy that involves investigation and implementation of known drugs for treatment of new diseases. This method significantly reduces the costs required for establishing the safety of a drug and is associated with higher rates of drug approval. Ciclopirox olamine (CPX) is an antifungal agent that has been on the market since the 1970s. The mechanism of action is believed to include disrupting DNA repair and cell division signals. The compound is also believed to affect the membrane transfer system by interrupting the Na+ K+ ATPase. We have recently been looking at the anti-tumor properties of CPX. In preclinical studies, CPX has been shown to inhibit growth of cell lines for human breast carcinoma, bladder cancer and colon adenocarcinoma. Here, we present our preliminary results of the effects of CPX on esophageal carcinoma cells. 

Methods:  
We maintained two esophageal cancer cell lines, TE-7 and TE-10 in standard culture flasks with RPMI media. Cell lines were exposed to CPX and the effects are reported. Proliferation assays were performed as follows. Cultured cells from each cell line were exposed to CPX for 24, 48 and 72 h, after which cells were lysed and hexosaminidase assay was performed. Colony formation assays were performed as follows. Cultured cells were diluted to 500 cells/mL, plated, and exposed to increasing concentrations of CPX for 48 h. After exposure, cells were incubated in standard media for 10 days. Cells were then fixed and stained with crystal violet dye. Cell cycle analysis was performed on cells from both cells lines after exposure to CPX for 48 h. Cells were permeabilised and treated with propidium iodide before running through flow cytometry. 

Results:
CPX causes growth inhibition of TE-7 and TE-10 esophageal cancer cell lines. Results of proliferation assay revealed growth inhibition of esophageal cancer cells from TE-7 and TE-10 cell lines in a dose and time-dependent manner. Cell cycle assay also showed growth inhibition of both cell lines in a concentration dependent manner. On cell cycle analysis, cells exposed to CPX revealed cell-cycle arrest in G2/M phase

Conclusion:

Ciclopirox olamine (CPX) is an FDA-approved antifungal agent recently shown to have anti-tumor properties. To date, there is no published data on CPX as a potential target of esophageal carcinoma.  We have demonstrated growth inhibition of two esophageal cancer cell lines when exposed to CPX. These data pave the way for further research into the mechanisms of growth inhibition and potential use of this agent in the treatment of esophageal carcinoma.

42.18 Reverse Thermal Gel: A Candidate For An Early Minimally Invasive Method To Patch Myelomeningoceles

Posted on January 25, 2017

J. R. Bardill1, D. Park1, S. Williams3, A. Marwan1,2,3  3University Of Colorado Denver,Pediatric Surgery,Aurora, CO, USA 1University Of Colorado Denver,Bioengineering,Aurora, CO, USA 2Children’s Hospital Colorado,Aurora, CO, USA

Introduction:   Neural tube defects (NTDs) result in lifelong neurological and functional deficits.  Currently, an in-utero surgical repair of NTDs covers/patches the defect, resulting in a significant 50% reduction in post-natal ventriculoperitoneal shunting. However, this surgery is complicated by risks of morbidity to the mother and infant.  Studies have found that early in gestation, failed neural tube closure results in progressive tissue damage secondary to exposure to amniotic fluid.  Therefore, the goal of current NTD repair is to cover the defect at an earlier gestational age to prevent the irreversible damage to neural tissue from taking effect.  The development of a novel, biomimetic, Reverse Thermal Gel (RTG) has posed a unique solution to deliver an early, minimally invasive method to patch a NTD.  Here, we demonstrate two in-vitro properties of the RTG for NTD applicability:  1.) Fast in-situ gelling properties in amniotic fluids, 2.) Gel stability after 1 month of exposure to amniotic fluid.

Methods:   RTG gelling stability and lower critical solution temperature (LCST) testing in amniotic fluids:  In glass vials, RTGs were dissolved in phosphate buffered saline (PBS), sheep amniotic fluid (SAF), and human amniotic fluid (HAF) and gelled in a warm water bath.  Stable gels were assessed using the vial tilt test and the LCST was determined using temperature sensitive UV-VIS spectroscopy.  B.)  RTG 1 month degradation:  RTGs were exposed to PBS, SAF, and HAF for 1 month and the LCST was tested to determine if RTG properties had changed.

Results:  The RTG shows stability when gelled in amniotic fluids and an LCST below physiologic temperature, which is necessary for in-situ gelling properties for this NTD application.  After 1 month of exposure to amniotic fluids, the LCST remained below physiologic temperatures, indicating the RTG will remain stable for 1 month in the amniotic environment.

Conclusion:  The RTG has fast in-situ gelling properties in amniotic fluid that remain stable over time, making it an ideal candidate for an early, minimally invasive method to patch NTDs.

 

42.17 The Rapamycin Analog Temsirolimus Ameliorates Systemic Inflammation after Cecal Ligation and Puncture in Mice

Posted on January 25, 2017

A. J. Lewis1, X. Zhang1, M. R. Rosengart1,2  1University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh,Department Of Critical Care Medicine,Pittsburgh, PA, USA

Introduction:
Temsirolimus is a rapamycin analog shown previously by our group to induce autophagy and decrease acute kidney injury (AKI) during endotoxemia in both pre- and post-treatment studies.  However, the degree to which it functions as a therapeutic to improve the outcome during established sepsis remains to be determined. Cecal ligation and puncture (CLP) generates a systemic inflammatory response from three insults: surgical tissue trauma, ischemia from the ligated cecum, and host barrier breakdown from cecal puncture; it is considered a physiologically relevant model of surgical sepsis.  We hypothesize that after CLP, treatment with temsirolimus will attenuate the host inflammatory response to septic insult and decrease organ injury.

Methods:
Male C57BL/6 mice (8-12 weeks) underwent 1cm ligation, double 21-gauge puncture CLP using ketamine/xylazine anesthesia.  No antibiotics were administered.  One hour after CLP, mice were randomized to receive a 5 mg/kg intraperitoneal injection of temsirolimus or equivalent volume of diluent.  Mice were monitored for 24 hours, euthanized, and plasma and tissue were harvested.  Plasma cytokine and Cystatin C concentrations were assayed via ELISA.  Alanine aminotransferase (ALT) concentration was determined using a Heska Dri-Chem automated analyzer.  Statistical significance (p<0.05) was determined by rank sum.

Results:
When compared to controls, mice receiving temsirolimus exhibited significantly lower concentrations of IL-6 (1253 vs. 32695 pg/mL, p=0.033), TNF-a (36.4 vs. 176.4 pg/mL, p=0.049), and IL-10 (1033 vs. 4146 pg/mL, p=0.017).  Cystatin C, a sensitive marker of AKI, was lower in temsirolimus-treated animals (868 vs. 1392 ng/mL, p=0.158).  We found no difference in liver injury, as quantified by ALT, between groups (119 vs. 98 U/L, p=0.322).

Conclusion:
Temsirolimus treatment after CLP sepsis reduces systemic inflammation. This reduced inflammation correlated with attenuated AKI, though this did not attain statistical significance. These data suggest that potential exists for the use of temsirolimus as a therapy to reduce deleterious effects of a hyperinflammatory state of sepsis.  Further experiments are needed to determine the effects of temsirolimus on host physiology and survival.
 

42.16 Overexpression of DYRK2 reduces viability of pancreatic cancer cells by activation of GSK3β signaling

Posted on January 25, 2017

T. Horiuchi1,2, H. Shiba1, N. Saito1,2, H. Sugano1,2, Y. Shirai1, R. Iwase1, K. Haruki1, Y. Fujiwara1, R. Mimoto1,3, K. Furukawa1, T. Uwagawa1,4, K. Yoshida3, T. Ohashi2, K. Yanaga1  1The Jikei University School Of Medicine,Department Of Surgery,Tokyo, , Japan 2The Jikei University School Of Medicine,Department Of Gene Therapy, Research Center For Medical Science,Tokyo, , Japan 3The Jikei University School Of Medicine,Department Of Biochemistry,Tokyo, , Japan 4The Jikei University School Of Medicine,Department Of Medical Oncology And Hematology,Tokyo, , Japan

Introduction: Pancreatic cancer is one of the most aggressive cancer and the fourth leading cause of cancer-related death in the United States and fifth in Japan. The standard first-line treatment for the patients with unresectable locally advanced or metastatic pancreatic ductal adenocarcinoma has been gemcitabine therapy. However, the median survival time did not exceed 6.5 months. Thus, it is imperative to identify the molecular mechanism of pancreatic cancer progression in order to develop novel-targeted therapies to improve the overall survival of the patients. DYRK2 is a member of dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) family that autophosphorylate tyrosine residues in their activation-loop and function as serine/threonine kinases on their substrates. It is reported that DYRK2 directly phosphorylates p53 at Ser46 and induces apoptosis in response to DNA damage. Moreover, the down-regulation of DYRK2 contributes to accelerated proliferation by stabilizing c-Jun and c-Myc. In this study, we assessed the effects of DYRK2 overexpression on the regulation of pancreatic cancer cell viability.

Methods: Flag-tagged DYRK2 was cloned into the pEB multi vector and transfected into human pancreatic cancer cell line, PANC-1. We compared DYRK2 overexpression group with empty vector-transfected group or non-treat group as control. To assess cell viability and examine the underlying molecular events, we performed cell viability MTT assay and Western blot analysis.

Results: DYRK2 expression was markedly decreased in PANC-1 cells. Cell viability in DYRK2 overexpression group was significantly lower than that in empty vector-transfected group (p=0.023). DYRK2 overexpression upregulated the phosphorylation of glycogen synthase kinase (GSK)-3β protein and reduced cyclin D1 expression in PANC-1 cells.

Conclusion: DYRK2 overexpression activated GSK-3β signaling and significantly reduced cell viability of human pancreatic cancer cells.

 

42.15 Oridonin Enhances the Anti-inflammatory Effects of Heat Shock Protein 70 Inhibition in Hepatic Stellate Cells

Posted on January 25, 2017

O. Nunez Lopez1, X. Wang1, F. J. Bohanon1, H. Tie1, G. Graham1, R. S. Radhakrishnan1  1University Of Texas Medical Branch,Surgery,Galveston, TX, USA

Introduction:
Activated hepatic stellate cells (HSCs) are the main extracellular matrix (ECM) producers in liver fibrosis and play an important role in hepatic inflammation. Their inactivation and induction of apoptosis have become an important therapeutic strategy. HSP70 inhibitors have been reported to exert anti-proliferative effects in cancerous cells. However, little is known about the effects of HSP70 inhibition on hepatic fibrosis. Previously, our group reported that Oridonin, a plant-derived compound, has anti-inflammatory properties in HSCs via the NF-kB signaling pathway. In the present study, we determined the role of HSP70 inhibition alone and in combination with oridonin on the LPS-induced hepatic fibrogenic response in HSCs.

Methods:
The human activated HSC line LX-2 was used for this study. Cytokines production was measured by ELISA. Cellular proteins were analyzed by Western immunoblotting and immunofluorescence staining.

Results:
PES-CI (HSP70 inhibitor) treatment decreased the production of the LPS-induced pro-inflammatory cytokines IL-6 and MCP-1 in a dose-dependent manner in LX-2 cells. Moreover, micromolar concentrations of PES-CI (2.5µM) and Oridonin (2.5 µM) synergistically suppressed LPS-induced IL-6 and MCP-1 secretion. Compared to treatment with either PES-CI or oridonin alone, combined treatment (PES-CI + oridonin) resulted in significantly lower LPS-induced IKKβ and IκBα phosphorylation and lower LPS-induced NF-kBp65 nuclear translocation and DNA binding.

Conclusion:
Oridonin acts synergistically with the HSP70 inhibitor PES-CI. Combined treatment has anti-inflammatory properties via the NF-kB signaling pathway.
 

42.14 Reverse Thermal Gel: An Alternative To Fetal Repair Of Myelomeningocele III: In Utero Applicability

Posted on January 25, 2017

A. Marwan1, S. Williams1, J. Bardill2, N. AbdulAziz1, D. Park2, A. Marwan1  1University Of Colorado School Of Medicine,Pediatric Surgery,Aurora, CO, USA 2University Of Colorado School Of Medicine,Bioengineering,Aurora, CO, USA

Introduction:  Myelomeningocele (MMC) is the leading cause of central nervous system malformation in humans and a devastating birth defect resulting in destruction of the developing spinal cord. Prenatal closure of MMCs occurs at 22-26 weeks gestation and results in a 50% reduction in the need for postnatal ventriculoperitoneal shunting. However, it is associated with significant morbidities including premature rupture of membranes, preterm labor, and uterine scar dehiscence. Our lab focuses on the development of a minimally invasive bioengineering alternative to open fetal repairs. Previously, we demonstrated that the reverse thermal gel (RTG) is stable in amniotic fluid, has negligible permeability, possesses a unique ultrastructure and supports viability and proliferation of murine neonatal fibroblasts and keratinocytes. However, the in utero applicability of the RTG is currently unknown. This abstract focuses on the effects of in vivo polymer injection. 

Methods: Ex-utero embryo culture: A time pregnant E14.5 female mouse with an open MMC was anesthetized and the uterus explanted in media. The Embryo was separated from maternal decidua with an intact visceral yolk sac and placed in rat serum. Culture tubes were filled with 5% carbon dioxide, 10% oxygen, and 85% nitrogen mix and incubated. Embryos were recovered for 1-hour. Microinjection of the RTG was performed directly onto the caudal region of the embryo. After injection, embryos were re-gassed and returned to the incubator. Embryos were cultured for 8 (short culture) and 15 hours (long culture). In Utero applicability: 3, 10 and 20ul of either polymer or saline was injected with or without ultrasound (US) guidance into the amniotic sac of E13.5 pregnant curly tail or wild type mice after delivery of the uterus. A total of 19 fetuses were injected. Animals were recovered and examined daily for preterm labor. Technical success evaluated by identification of embryos with a phenotype, successful delivery of the RTG, adequate polymer gelling and presence of the polymer at the time of delivery. Fetal survival was assessed by echocardiography prior to cesarean section. 

Results:Direct microinjection of the RTG through the yolk sac does not negatively affect viability of the embryo, and is associated with yolk sac vascularity and further embryonic development as evidenced by otic vesicle formation. We appropriately identified embryos with a curly tail phenotype. Ultrasound guided In-Utero injection of the polymer is technically successful, accompanied by appropriate polymer gelling without disruption by amniotic fluid dynamics and is associated with fetal survival. Upon cesarean section, the polymer is identified at the initial site of injection.

Conclusion:Our novel RTG polymer demonstrates physical and biological properties conducive to its use as a potential alternative to fetal closure of MMCs. Current research is focused on evaluating the effects of RTG as an alternative MMC closure mechanism. 

 

42.13 Thailandepsin-A Decreases Notch1 and Cell Proliferation in Neuroblastoma

Posted on January 25, 2017

Z. Aburjania1, A. Janssen1, S. Jang1, H. Chen1, R. Jaskula-Sztul1  1The University Of Alabama At Birmingham,Surgery,Birmingham, Alabama, USA

Introduction:
Neuroblastoma is a highly aggressive tumor of childhood with 5year survival rate of 68% for children aged 1 to 14 years. It is derived from sympathoadrenal lineage of the neural crest progenitor cells. Notch signaling has important role in determining cell fate during differentiation of ectodermal cells in the sympathetic nervous system. Notch pathway is associated with low differentiation of neuroblastoma, activation of proliferation and motility of cancer cells. Histone deacetylase inhibitors (HDACi) are well-known for their anti-tumor activity as well as their ability to regulate Notch signaling.  We tested a novel HDACi Thailandepsin-A (TDP-A) as a treatment option for neuroblastoma.

Methods:
Methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay was used to determine the IC50 of TDP-A on LA-N-5, NGP and SK-N-SH neuroblastoma cell lines. The effect of TDP-A on Notch1 and apoptotic markers p21 and p27 were assessed by western blot after treatment of neuroblastoma cells with DMSO, 3nM and 6nM TDP-A. Additionally N-myc-amplified NGP cell lysates were assessed for N-myc protein levels. Cells were also assessed for neurite outgrowth as a sign of differentiation.

Results:
IC50 was determined to be 4nM, 4nM and 6nM for LA-N-5, NGP and SK-N-SH cell lines respectively. Western blot analysis showed decrease of Notch1 in all cell lines. p27 levels increased with the treatment in LA-N-5 cells. NGP cell line showed increase of p21 protein. N-myc levels decreased in NGP cell lines with the treatment of TDP-A. Increase of neurite outgrowth was observed with the treatment in LA-N-5 cells (12.5um vs 47.8um, p=0.464).

Conclusion:
TDPA has potent anti-proliferating effect on neuroblastoma cell lines in low nanomolar concentrations. It decreased Notch1 in all cell lines together with decrease of N-myc in NGP that is known to have amplified N-myc gene, which is observed in one-third of neuroblastomas and correlated with advanced disease. These results show that TDPA is a promising drug for treatment of neuroblastoma.
 

42.12 Progenitor Cell Therapies for Traumatic Brain Injury: a Meta-Analysis of Pre-Clinical Data

Posted on January 25, 2017

M. L. Jackson1, A. K. Srivastava1, C. S. Cox1  1McGovern Medical School,Department Of Pediatric Surgery,Houston, TX, USA

Introduction: There is currently no treatment available to reverse the primary injury associated with traumatic brain injury (TBI). Progenitor cell therapies have shown promise in both pre-clinical and clinical studies. We conducted a meta-analysis of pre-clinical studies using progenitor cells for the treatment of TBI. 

Methods: PubMed, Cochrane Review, and Google Scholar were searched for articles using mesenchymal stromal/stem cells, umbilical cord derived stem cells/cord blood, placental stem cells, amniotic fluid derived stem cells, neural stem cells, and TBI. Inclusion criteria were (1) animal model of TBI with (2) administration of stem/progenitor cells, (3) and outcome measures that included either brain lesion volume (LV) assessment or rotarod (RR) motor function assessment, or both. Studies were excluded if they were not available in English, or if their design included combination with non-cellular therapies. Two investigators independently performed literature searches and screened abstracts. Discrepancies were decided by a third investigator. Studies meeting inclusion criteria underwent data extraction. Analysis was performed using Review Manager 5.3 according to a random-effects model; values are expressed as standard mean differences with 95% confidence interval (CI). All studies underwent quality scoring based on a combination of the recommendations for transparent reporting of basic science research from the NINDS, and a quality score that was developed for grading pre-clinical studies in stroke.

Results: Of 456 abstracts identified, 55 met inclusion criteria and underwent full text evaluation. Twelve studies evaluated RR testing as an outcome measure, and eight reported data that could be extracted. Nineteen studies evaluated LV, and sixteen reported data that could be extracted. Average quality score for the eight RR studies was 3.6 ± 0.7, and LV was 4.5 ± 1.8 out of 8 possible points. No study achieved a perfect score. LV outcomes favored cell treatment with standardized mean reduction of 1.43 (95%CI 0.85-2.00). RR outcomes showed a trend towards standard mean improvement of 0.59 which was not statistically significant (95%CI -0.20-1.37). Heterogeneity (I2) was 84% for LV analysis and 74% for RR analysis, indicating a high amount of heterogeneity among the studies compared.

Conclusions: Our meta-analysis evaluates both histological and functional outcome of progenitor cell therapies for TBI, and reports that despite heterogeneity among the studies there is a benefit in lesion volume reduction, and a trend towards overall improvement in motor function.

42.11 Direct Peritoneal Resuscitation Improves Mesenteric Perfusion by Nitric Oxide Dependent Pathways

Posted on January 25, 2017

S. Khaneki1, A. Jensen1, N. Drucker1, T. Markel1  1Indiana University School Of Medicine,Surgery,Indianapolis, IN, USA

Introduction: Direct peritoneal resuscitation (DPR) has been shown to increase survival following intestinal ischemia and reperfusion injury (I/R).  We have previously appreciated that the use of minimal essential medium (MEM) contributes to these survival benefits as well as a reduction in intestinal inflammation. We hypothesized that: 1) DPR using MEM as a dialysate would increase mesenteric perfusion following intestinal I/R, and 2) increased perfusion would be dependent on endothelial nitric oxide pathways.  

Methods: Eight-week-old C57Bl6J wild type (WT) and C57Bl6J eNOS Knock Out (eNOS KO) male mice were anesthetized with isoflurane.  A midline laparotomy was performed and the intestines were eviscerated. Baseline perfusion was measured using Laser Doppler Imaging (LDI).  Intestinal ischemia was then induced by clamping the superior mesenteric artery (SMA) with a non-traumatic vascular clamp for 60 minutes.   Following intestinal ischemia, the clamp was removed and the incision was closed in two layers.  Prior to final closure, 1ml of PBS vehicle or 1 ml of MEM (alpha-minimum essential medium plus 16% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine) was injected into the abdominal cavity of the WT and eNOS KO groups. Animals were then allowed to recover for 48 hours.  After this time, animals were reanesthetized, the midline incision opened, and intestinal perfusion reassessed by LDI.  Animals were then euthanized.  Perfusion was expressed as a percentage of baseline and was compared between groups using the Mann-Whitney test.  A p<0.05 was considered statistically significant.

Results:Direct peritoneal resuscitation with MEM significantly improved mesenteric perfusion compared to PBS as measured by laser doppler imaging (WT + Vehicle: 32.85±17.2% vs. WT + MEM: 105.9±6.58%, p<0.05).  The benefits of DPR with MEM were lost when endothelial nitric oxide signaling pathways were ablated (WT + MEM: 105.9±6.58% vs. eNOS KO + MEM: 19.65 ± 5.49%, p<0.05).

Conclusion:Direct peritoneal resuscitation with MEM has significant therapeutic potential for its ability to improve mesenteric perfusion following intestinal ischemia. This benefit appears to be dependent on nitric oxide signaling within the mesenteric endothelium.  Further investigation into additional downstream signaling cascades are essential prior to DPR being implemented in human clinical trials.

 

42.10 Gabapentin Attenuates Whisker Hypersensitivity After Diffuse Traumatic Brain Injury in the Rat

Posted on January 25, 2017

S. Ogle1,2,3, L. M. Law1,2, P. D. Adelson1,2, J. Lifshitz1,2, T. C. Thomas1,2,3, S. B. Johnson1,3  1University Of Arizona,College Of Medicine, Child Health,Phoenix, AZ, USA 2BARROW Neurological Institute @ Phoenix Children’s Hospital,Translational Neurotrauma Research Program,Phoenix, AZ, USA 3Banner University Medical Center- Phoenix,Surgery,Phoenix, AZ, USA

Introduction:  Chronic neurological dysfunction occurs in 15-20% of mild diffuse traumatic brain injury (TBI) patients have been highlighted in athletic and military populations. Similar to light and sound hypersensitivity after TBI in humans, rodents develop a late-onset, gain-of-function hypersensitivity to whisker stimulation at 28 days following experimental diffuse TBI. Change in sensory sensitivity manifests, in part, as a consequence of injury-induced maladaptive circuit reorganization. Mechanistically, maladaptive circuit reorganization after TBI is not fully understood; however, bursts of new synapse formation can occur via astrocyte secreted thrombospondin-1 (TSP-1) interactions with α2δ-1-subunit on voltage-gated calcium channels. Preliminary data demonstrate an injury-induced increase in TSP-1 in the somatosensory relays of the whisker circuit. In seizure and focal injury models, gabapentin (GBP) inhibits TSP-1/ α2δ-1 interactions, inhibiting synaptogenesis and consequently attenuating morbidity, however this has never been addressed in diffuse TBI. Thus, we hypothesize that gabapentin therapy after experimental diffuse TBI will attenuate whisker hypersensitivity in a rodent model of diffuse TBI. 

Methods: Adult male Sprague-Dawley rats underwent diffuse TBI by moderate midline fluid percussion brain injury. Vehicle (0.9% normal saline) or GBP (100mg/kg/day, dissolved in 0.9% normal saline) was delivered continuously via subcutaneous osmotic pumps. Animals were randomly assigned one of the following groups (n=12-15/group): naïve+vehicle, naïve+GBP, injured+vehicle, injured+GBP.  Pumps were placed immediately after the injury procedure and continued for a total of 14 days post- injury (DPI). Whisker sensitivity was quantified via the whisker nuisance task (WNT) at 28 DPI. A Kruskal-Wallis non-parametric test followed by a Dunn’s multiple comparison post-test was used for statistical analysis of WNT scores. 

Results:At 28 DPI, there were no significant differences between naïve+vehicle and naïve+GBP animals (p=0.95), thus these animals were combined for analysis. WNT scores significantly changed in response to injury (KW=11.36), where WNT scores in injured+vehicle rats were significantly greater than naïve rats (p<0.01), as previously reported. WNT scores of injured+GBP rats were not statistically different from naïve animals, albeit they were not significantly lower than the WNT scores of injured+vehicle. 

Conclusion:These preliminary data indicate that treatment with gabapentin in the peri-traumatic period results in WNT scores statistically similar to naive animals, suggesting a prophylactic intervention for sensory hypersensitivity. Future studies necessitate a dose response evaluation and confirmation of TSP-1/ α2δ-1 mechanisms of action. Acute bolus gabapentin administration may be a FDA-approved approach to mitigate chronic neurological dysfunction after TBI. 
 

42.09 High-performance Machine Learning and Evolutionary Computing to Develop Personalized Therapeutics

Posted on January 25, 2017

R. C. Cockrell1, G. An1  1University Of Chicago,Surgery,Chicago, IL, USA

Introduction: Personalized medicine requires the right interventions for the right patient at the right time. This necessitates parsing individual patient trajectories at a mechanistically relevant temporal resolution, a task for which existing biomedical data sets are inadequate. High-performance computational modeling and simulation can help dynamically contextualize multi-dimensional data arising from complex systems; however, knowledge of the mechanics of a complex system does not directly lead to the understanding of how to alter these mechanics to a specific end. The combination of high performance simulation and machine learning methods provides a means to identify sets of putative interventions tailored to specific system trajectories. In this study, we examine and assess the efficacy of two methods of developing control strategies for a stochastic dynamical immune system: evolutionary algorithms and artificial neural networks.

Methods: All experiments were performed using a previously validated agent-based model of systemic inflammatory response syndrome.  Complete chemokine data for neural net forecasting was collected every seven simulated minutes for approximately 150,000 in silico patients.  This data was used to train a convolutional “deep-learning” neural network to determine patient state at a future point in time using a five-point scale.  In order to develop interventions using evolutionary algorithms, each intervention was represented as a numerical vector of protein-synthesis augmentations and inhibitions. The genetic algorithm would run until the starting population of interventions converged to a single or small number of near-optimally fit interventions.  Intermediate data was saved and used to train a convolutional neural network that produces a near-optimal intervention vector as output.

Results:We show that neural network forecasting models can be used to accurately predict in silico patient disease progression with sufficient data sampling when considering the immune system to be a stochastic dynamical system.  Additionally, we show that both evolutionary algorithms and artificial neural networks can be used develop treatment strategies for in silico patients suffering from sepsis.  Evolutionary algorithms are directed primarily by characteristics if disease (invasiveness, toxigenesis, virulence, etc.) while artificial neural networks operate on patient state (serum levels of chemokine molecules and their associated rates of change).  While both methods show the ability to improve patient state and probability of a positive outcome, this study confirms the conventional wisdom that optimal outcomes can be most widely achieved with personalized solutions.

Conclusion:Machine learning combined with computational simulation of disease provides a viable path towards personalized medicine.  Artificial neural networks represent the most efficient strategy to develop interventions that guide the immune system to a desired state.

42.08 Human Necrotizing Enterocolitis (NEC) Genome Analysis Shows Relaxin Family Gene Changes

Posted on January 25, 2017

C. Kapsalis1, P. J. Matheson1, J. Hata2, J. Shepherd1, L. M. Bond1, J. W. Smith1, C. D. Downard1  1University Of Louisville,Surgery,Louisville, KY, USA 2Kosair Children’s Hospital,Pathology,Louisville, KY, USA

Introduction:  NEC affects premature infants and is a leading gastrointestinal cause of infant death. Formula-fed infants have a 6 to 10X greater risk of developing NEC compared to breast fed infants.  Relaxin, a hormone of pregnancy found at high concentrations in maternal milk compared to that of blood, might play an important role in preventing the intestinal sequelae in patients at risk for NEC.  Thus, decreased expression of relaxin-family receptor (RXFP) or relaxin protein (RLN) might be a critical event in the development of the disease.  We hypothesized that gene expression of gut RXFP might be decreased in infants with clinically diagnosed NEC.

Methods:  IRB approval was obtained to collect paraffin-embedded gut tissue samples from patients with pathologist-graded NEC and Controls.  All study personal were blinded to patient groups.  Qiagen RNeasy FFPE was performed to isolate total RNA from tissue samples.  mRNA was quantified and analyzed for Quality Control (QC).  Total cDNA was obtained using a NuGen Ovation RNA Amplification V2 kit.  Biotin-labeled aRNA was created for analysis on an Illumina HumanHT-12 v4 BeadChip to measure the entire human genome in each sample.  Ingenuity Pathway software was used to analyze the results.

Results: Twenty-four (24) tissue samples were obtained and processed yielding an average of 1,230 ng mRNA per sample.  QC demonstrated a 5S marker peak, which is consistent with mRNA extracted from paraffin-embedded samples.  The Illumina HumanHT-12 v4 BeadChip demonstrated increased relaxin 4 gene expression (1.54x compared to Control*P<0.05).  Other relaxin receptor and relaxin isoforms were not significantly different.

Conclusion: QC and mRNA levels from FFPE extraction were consistent with expected results and biotin-labeled RNA for the BeadChip was sufficient for the genome evaluation.  Ingenuity Pathway analysis showed RXFP-4 upregulation suggesting altered relaxin family gene expression in NEC.  We have previously shown that RXFP protein levels are altered as early as 48 hours prior to histologic changes of NEC.  These results support those prior animal studies.

 

42.07 Up Regulation of Somatostatin Receptor Expression on Medullary Thyroid Cancers Using HDACi

Posted on January 25, 2017

A. Janssen1, Z. Sun1, Z. Aburjania1, H. Jin1, R. Jaskula-Sztul1, H. Chen1  1University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA

Introduction: Medullary Thyroid Cancers (MTC) are neuroendocrine tumors (NETs) that arise from calcitonin-producing C-cells of the thyroid gland. Although MTCs are slow growing, they are frequently metastatic at the time of discovery and not amenable to curative surgery. They also secrete excessive hormones, often causing debilitating symptoms such as facial flushing and diarrhea. Somatostatin receptors (SSTRs) are a family of five G protein-coupled receptors that are overexpressed in 85% of NETs. SSTRs participate in modulation of neurotransmission, inhibition of hormone secretion, and inhibition of cell proliferation. Somatostatin (SST) analogs have been used for treatment and imaging studies in NETs, but have not shown to be very practical in MTCs due to low or lack of SSTR expression. Herein, we describe the novel use of a class of drugs known as histone deacetylase inhibitors (HDACi) that have the unique ability to up regulate expression of SSTRs on MTCs, and are able to induce expression in patients with no phenotypic expression of SSTRs. The use of HDACi will further advance SST analog therapies and imaging studies in NETs.

Methods: We used Quantitative real time PCR (Q-PCR) to evaluate basal expression of SSTR1 to SSTR5 on two MTC cell lines– MZ and TT– against a positive control pulmonary fibroblast cell WI-38. Time dependent induction of SSTRs was then evaluated with Q-PCR for MZ and TT cells treated with a potent HDACi Thilandepsin-A (TDP-A) at 12, 24, and 48 hours. Furthermore, we assessed dose dependent induction for SSTR1-5 on MZ and TT at 48 hours with four separate HDACi– FK228, SAHA, TDP-A and VPA– using Q-PCR and Western Blot analysis for all treatments.

Results: Basal expression for both cell lines showed an increased expression in one or more SSTRs. MZ cells showed increased expression in SSTR2, SSTR4 and SSTR5, with very low expression of SSTR1. TT cells showed a significant expression of SSTR1 and SSTR2 with no basal expression of SSTR4. After Treatment with all four HDACi, MZ cells showed an eight- to fourteen-fold increase in message for SSTR1 and a two-fold increase in SSTR2. TT cells showed increased expression of SSTR2, SSTR3 and SSTR5. More importantly, TT cells had no basal expression of SSTR4, but treatment showed to induce expression of SSTR4.

Conclusion: We demonstrated that four tested HDACi increased message and protein levels of SSTRs in MTC cells in a time and dose dependent manner. We also showed that HDACi have the capability to induce expression of SSTRs even if there is no basal expression. This novel finding provides an avenue to improve SST analog therapies and imaging studies for MTCs.

42.06 Inhaled IL10 Decreases the Development of Pulmonary Hypertension in Slit3 Knockout Mice with CDH

Posted on January 25, 2017

M. Shah1, M. R. Phillips1, S. Balaji2, S. G. Keswani2, S. E. McLean1  1University Of North Carolina At Chapel Hill,Chapel Hill, NC, USA 2Baylor College Of Medicine,Houston, TX, USA

Introduction:
Congenital diaphragmatic hernia (CDH) is associated with pulmonary arterial hypertension (PAH). There is an increase in inflammatory cell invasion in pulmonary arteries in CDH. Slit3 knockout (KO) mice with CDH develop PAH over time, with no evidence of PAH at 2 weeks of age, but presence of PAH at 4 weeks of age. We hypothesize that treatment of 2 week old Slit3 KO mice with CDH that have yet to develop PAH, with inhaled IL10, an anti-inflammatory cytokine, will decrease the development of PAH in CDH.

Methods:
Slit3 wild-type (WT) (n=5) and KO (n=5) mice were analyzed at 2 weeks of age. Mice were treated with IL10 mixed with hydrogel, administered intranasally, at day 0 and day 7. Echocardiography was performed pre-IL10 (day 0) at 2 weeks of age and post-IL10 treatment (day 14) at 4 weeks of age. Right ventricular (RV) systolic pressures were obtained by direct cardiac puncture post-IL10 treatment (day 14) at 4 weeks of age. 

Results:
Pre-IL10 treatment, the pulmonary artery (PA) flow of KO mice was similar to WT, with PA velocity time indices (VTI), 20.95 mm vs. 21.80 mm (p=0.49), PA peak velocities, 546.59 mm/s vs. 547.86 mm/s (p=0.97), and pulmonary acceleration times (PAT), 11.1 ms vs. 12.22 ms (p=0.12), respectively. Post-IL10 treatment at 4 weeks of age, there was still similar PA flow in KO and WT mice, with PA VTI, 26.19 mm vs. 24.05mm (p=0.34), and PAT, 11.72 ms vs. 11.28 ms (p=0.67). PA peak velocity was altered in KO vs WT mice, however, 809.7 mm/s vs. 667.48 mm/s, respectively (p<0.05). Post-IL10 treatment, RV systolic pressure was similar in KO (n=3) and WT (n=4) mice, 19.38 mmHg vs. 18.22 mmHg, respectively (p=0.54).

Conclusion:
2 week old Slit3 KO mice with CDH that have yet to develop PAH show decreased development of PAH and maintenance of RV systolic pressures at 4 weeks of age after treatment with inhaled IL10. IL10 may play an anti-inflammatory role that ameliorates PAH in CDH.
 

42.05 Histologic Changes Following Fish Oil Pretreatment and Hepatic Ischemia Reperfusion Injury

Posted on January 25, 2017

M. A. Baker1, P. Nandivada1, A. Pan1, G. L. Fell1, L. Anez-Bustillos1, D. T. Dao1, K. M. Gura2, V. Nosé3, M. Puder1  1Boston Children’s Hospital,Vascular Biology Program And Department Of Surgery,Boston, MA, USA 2Boston Children’s Hospital,Department Of Pharmacy,Boston, MA, USA 3Massachusetts General Hospital,Department Of Pathology,Boston, MA, USA

Introduction:  Ischemia reperfusion injury (IRI) is a major barrier to liver surgery and transplantation, particularly with steatotic livers. Fish oil lipid emulsions are rich in anti-inflammatory omega-3 fatty acids and the antioxidant alpha-tocopherol. The purpose of this study was to determine if pre-treatment with a single dose of intravenous fish oil (IVFO) can decrease hepatic IRI.

Methods:  Male 6-8 wk old C57BL/6 mice received 1 g/kg IVFO (Omegaven®, Fresenius Kabi) or isovolumetric saline via tail vein injection 1 h prior to suture ligature of the portal triad to the cephalad liver lobes. After 30 min of 70% hepatic ischemia, livers were reperfused. Animals were sacrificed after 4, 8, or 24 h of reperfusion and livers harvested for histologic analysis by a single, blinded, board-certified pathologist. Percent necrosis was calculated using ImageJ software (NIH).

Results: After 4 h of reperfusion, saline-treated livers demonstrated marked ischemia around central veins diffusely, while IVFO-treated livers demonstrated small, scattered foci of necrosis with normal liver between necrotic areas. After 8 h of reperfusion, both saline and IVFO-treated livers demonstrated pale areas of cell loss with surrounding regenerating hepatocytes. There were more regenerating cells around areas of necrosis in IVFO-treated livers, which was confirmed with Ki67 staining and may represent faster recovery. After 24 h of reperfusion, both saline and IVFO-treated livers demonstrated patchy areas of frank necrosis and normal appearing liver, without preference for central vein or portal space, with a decreased overall percent area of necrosis in IVFO-treated livers (saline mean 9.27 ± SEM 2.50 %, n=7 vs. IVFO mean 1.70 ± SEM 0.44 %, n=9; P=0.03). Figure 1 demonstrates representative H&E images of livers after IRI at 200x magnification (open arrows = ischemia, closed arrows = necrosis).

Conclusion: Temporal findings after hepatic IRI suggest IVFO’s hepatoprotective effects may be related to accelerating healing. Specific fatty acid receptor knockout studies are underway to determine the mechanism of this protection.

 

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