5.13 Better Fat Transfer: The Specific Choices that Optimize Lipoaspirate Integrit

Posted on December 22, 2014

D. A. Atashroo1, T. Wearda1, J. Raphel2, K. Paik1, E. Zielins1, G. Walmsley1, M. Hu1, K. Senarath-Yapa1, S. Menon1, A. Luan1, R. Tevlin1, D. Duscher1, Z. Maan1, G. Gurtner1, D. Wan1, S. Heilshorn2, M. T. Longaker1  2Stanford,Material Science,Stanford, CA, USA 1Stanford,Plastic Surgery,Stanford, CA, USA

Introduction:

Based on it’s abundant availability, ease of harvest, and biocompatability, fat grafting has grown tremendously in popularity.  Even so, it suffers from unpredictable outcomes.  At this point, little is known about how specific components in a fat grafting algorithm affect graft quality and survival.  In this study, we use rheology, the study of material flow and deformation, to quantify the effects that cannula diameter, length, and shape, flow rate, and extensional flow have on the integrity of fat. 

 

Methods:

Lipoaspirate was harvested from cosmetic surgery patients using suction assisted liposuction.  To prepare lipoaspirate for grafting, it was allowed to settle and then centrifuged and filtered to remove blood, oil, and debris.  To test the effects of cannula size and length, a syringe pump was used to inject fat at a controlled flow rate through various gauge (g) cannulas ranging from 8 to 25 cm in length.  All other variables were then held constant, and fat was injected at variable flow rates ranges from .1 ml/sec to greater than 2 ml/sec.  The effects of extensional flow, a rheological concept where pulling of a material causes it to deform in the absence of shear, was then tested with specially designed cannulas.  After injection, all processed samples were then tested in triplicate on a rheometer to measure their viscoelastic properties.  Fat grafts from representative groups were then placed into the scalps of CD1 athymic nude mice.  Volume of fat grafts was assessed by micro-CT at baseline and after 8 weeks when grafts were explanted, weighed, and sectioned for histology.  

 

Results:

In general fat grafts injected through larger, shorter, straight cannulas and at slower flow rates exhibited less shear thinning.   Importantly, clear breakpoints existed for these variables.  The storage modulus (G’) of fat processed with 14g and larger cannulas was significantly higher than for fat processed with smaller gauge cannulas (*p < .05).  Similarly, the 8 cm cannula group had a significantly greater G’ than the 25 cm cannula group (*p < .05).  The optimal flow rate was .5ml sec, and this group had a significantly improved integrity compared to the 1 ml/sec and faster groups (*p < .05).  Interestingly, the .1 ml/sec group, even though the slowest flow rate, tended to break down more than the .5 ml/sec group (p = .08).  In-vivo results comparing graft survival recapitulated the rheological findings in representative groups.  

 

Conclusions:

Surgeon choice of fat grafting algorithm has a significant impact on the final integrity of placed fat.  This study outlines specific alterations that surgeons may make to respect the damaging effects of shear forces and thereby optimize placement outcomes.  

 

5.15 Assessing ASC Regenerative Potential: Does Harvest Method Matter?

Posted on December 22, 2014

D. Atashroo1, E. Brett1, D. Duscher1, Z. N. Maan1, E. R. Zielins1, K. Paik1, A. Whittam1, M. Lin1, A. Luan1, A. McArdle1, C. Duldulao1, G. G. Walmsley1, M. S. Hu1, R. Tevlin1, D. C. Wan1, G. C. Gurtner1, M. T. Longaker1  1Stanford University,Hagey Laboratory For Pediatric Regenerative Medicine, Department Of Surgery, Plastic And Reconstructive Surgery Division,Palo Alto, CA, USA

Introduction:  Human adipose derived stromal cells (ASCs) have emerged as a vital component in tissue engineering.  Even though liposuction is the primary method of obtaining ASCs, little is known about the effects of different liposuction methods on the regenerative potential of harvested ASCs.  Despite the growing popularity of ultrasound assisted liposuction (UAL) because it increase the ease, speed, and safety of harvest, a paucity of evidence exists regarding the adipogenic and osteogenic differentiation potential of cells obtained in this way.  In this study, we evaluate the regenerative capacity of ASCs from third and fourth generation UAL compared to the industry standard suction assisted liposuction (SAL).

Methods:  Lipoaspirate was obtained from paired elective surgery patients using UAL and SAL, and harvested for ASCs.  A portion of the cells were then sorted using Fluorescence Activated Cell Sorting (FACS) based on an established progenitor surface marker profile (CD34+CD31-CD45-), and the yield of viable ASCs was compared.  An MTT assay was performed on plated cells to compare their proliferation potential.  Cultured cells were then differentiated towards adipogenic and osteogenic lineages.  Oil Red O and Alkaline Phosphatase staining was performed at Day 7 of differentiation; Alizarin Red staining was performed at Day 14.  RNA was harvested at Day 0, 7, and 14 for qRT-PCR analysis of gene expression.  Paired full thickness wounds were placed on the dorsum of CD1 athymic nude mice and then a hydrogel scaffold alone or seeded with 250k SAL or UAL ASCSs was placed in the wounds.  Tissue regeneration was assessed serially until complete wound closure.

Results: SAL and UAL demonstrated equivalent viable ASC yield on FACS and proliferative potential on MTT assay (*p > .05).  There was no significant difference in adipogenic staining by Oil Red O, or osteogenic staining by Alkaline Phosphatase or Alizarin Red (*p > .05).  Similarly qRT-PCR  demonstrated non statistically significant expression of multiple osteogenic and adipogenic genes (*p > .05).  Importantly, wound healing was significantly improved in both cell assisted groups compared to hydrogel alone (*p < .05); however, healing between SAL and UAL groups was equivalent (*p > .05).

Conclusion: UAL offers a similarly successful method compared to traditional SAL for obtaining ASCs for tissue engineering.  Surgeons and scientists alike benefit from this additional, and arguably better, option for obtaining ASCs for cell based therapies in regenerative medicine.
 

5.16 Adipose Derived Stem Cell-Mediated Enhancement of Fat Graft Retention is Dose Dependent

Posted on December 22, 2014

E. R. Zielins1, K. Paik1, D. A. Atashroo1, Z. N. Maan1, A. Luan1, S. M. Vistnes1, G. Walmsley1, K. Senarath-Yapa1, R. Tevlin1, T. Wearda1, O. Marecic1, M. T. Longaker1, D. C. Wan1  1Stanford University,Division Of Plastic And Reconstructive Surgery,Palo Alto, CA, USA

Introduction:
Known as cell-assisted lipotransfer (CAL), the addition of autologous adipose derived stem cells (ASCs) has shown much promise as a technique to improve fat graft take.  As the number of ASCs required for optimum fat graft retention remains unknown, we have attempted to determine this using a murine model of fat grafting.

Methods:
Human fat was mixed with ASCs obtained from patient-matched lipoaspirate and injected into the subcutaneous plane of the scalps of athymic nude mice.  The number of ASCs injected per 200μL fat graft ranged from 10,000 to 10 million.  Fat graft volume retention was assessed via CT scanning at 8 weeks, with explanted grafts compared histologically for overall architecture and vascularity.

Results:
Maximum fat graft retention was seen with the addition of 10,000 ASCs, with volumes significantly larger (*p<0.05) than those of unsupplemented grafts.  The addition of higher number of cells negatively impacted fat graft retention, with supplementation with 10 million ASCs producing the lowest final volumes.  Overall fat graft architecture and was not affected by supplementation with ASCs, though fat grafts supplemented with 10,000 ASCs showed increased vascularity.

Conclusion:
Our study has shown that there is in fact dose dependence in the number of ASCs that can be added to a fat graft.  While known for their pro-adipogenic and pro-angiogenic effects, it would seem that the ability of ASCs to enhance fat grafting must be counterbalanced by their potential to outcompete resident adipocytes and stromal cells for nutrients during the post-graft period.

5.17 Alteration in VEGF-A Expression Contributes To The Pathophysiology of Necrotizing Entercolitis (NEC)

Posted on December 22, 2014

J. A. Shepherd2, P. J. Matheson1,2, L. A. Galganski4, J. W. Smith2, R. N. Garrison1,2, C. D. Downard2  1Robley Rex Veterans Affairs Medical Center,Surgery,Louisville, KY, USA 2University Of Louisville,Hiram C. Polk Jr. M.D. Department Of Surgery,Louisville, KY, USA 4University Of California – Davis,Department Of Surgery,Sacramento, CA, USA

Introduction:

The pathophysiology of NEC impairs ileal blood flow via dysregulation of mediators of vascular tone, but deranged intestinal angiogenesis might also contribute to impaired blood flow. Vascular endothelial growth factor A (VEGFA) may have an important role in neonatal intestinal vascular development and control. We hypothesized that the gene and protein dysregulation that occurs during experimental NEC results in altered VEGFA protein expression.  

Methods:

Sprague-Dawley rats were randomized to groups by litter. CONTROLS were delivered vaginally and dam-fed. NEC groups were delivered by C-section 12 hrs prematurely, formula fed, exposed to intermittent cold and hypoxia, and given a single oral dose of lipopolysaccharide. Ileum samples were obtained at 0, 12, 24, 48, 72 and 96 hours of life and Western blots were performed with antibodies against VEGFA and β -actin for normalization by individual animal. Serum levels were analyzed for the 12, 24, 48, 72 and 96-hour time points by Luminex MagPix multianalyte cytokine array. Statistical analysis was performed using 2-way ANOVA and a priori P<0.05. 

Results:

Ileal VEGFA protein expression peaked at 12 hours in the CONTROLS. This peak was significantly decreased in the NEC compared to CONTROLS (6.62±0.66 vs. 2.01±0.36 in NEC, *P<0.05). The NEC groups had a peak in VEGFA expression at 96 hours of life, which was increased compared to CONTROLS (1.60±.14 vs. 0.61±0.05, *P<0.05). Serum levels for VEGFA expression peaked at 24 hours in CONTROLS. The NEC group expression was significantly decreased at this time point. The NEC group did experience a peak in serum VEGFA expression at 96 hours of life when compared with the CONTROL group.    

Conclusion:

This early peak in VEGFA expression suggests an important role for VEGFA in developmental angiogenesis in the CONTROLS that appears to be diminished in this experimental model of NEC. Further investigation of serum levels for VEGFA displayed the same concentration pattern, only delayed by 12 hours for each time point. These findings corroborate our prior studies that showed decreased expression of plate-derived growth factor (PDGF) and PDGF receptors. The deranged microvascular control of intestinal perfusion that occurs in NEC involves both altered mediators of vascular tone and mediators of vascular growth.

5.18 Relaxin Supplementation Affects Vascular Endothelial Growth Factor Expression in Experimental NEC

Posted on December 22, 2014

A. Clarke4, P. Matheson1,2, E. Stamper4, J. Smith2, R. N. Garrison1,2, C. D. Downard3  1Robley Rex Veterans Affairs Medical Center,Surgery,Louisville, KY, USA 2University Of Louisville,Surgery,Louisville, KY, USA 3University Of Louisville,Pediatric Surgery,Louisville, KY, USA 4University Of Louisville,School Of Medicine,Louisville, KY, USA

Introduction:  Necrotizing enterocolitis (NEC) is an intestinal disease which primarily affects premature infants.  Relaxin (RLXN), a hormone of pregnancy, dilates intestinal microvasculature and increases angiogenesis, possibly via the vascular endothelial growth factor (VEGF) family. VEGF-A/VEGF-R2 is the most studied angiogenic ligand/receptor interaction, while the other factors and receptors have been vastly less scrutinized. However, it has been shown that VEGF-R1 plays a mediating role for VEGF-A/R2 while also interacting with VEGF-B, an important factor in vascular survival. Oral relaxin supplementation has been shown to lessen the severity of NEC in rat models, and we hypothesize that this is due at least partially to the upregulation of VEGF and its receptors.

Methods:  NEC was induced in Sprague-Dawley rats by premature delivery, formula feeds, oral LPS, and episodic hypoxia/hypothermia.  Time-matched Controls (n=12) were vaginally delivered and dam fed.  NEC groups were randomized by litter (n=12/group): 1) NEC only, 2) NEC + 1x oral RLXN supplement (at 44 hours), and 3) NEC + oral RLXN supplement in All Feeds.  At 48 hours, ileum was recovered, mounted in paraffin, and either stained with H&E or left for Immunohistochemistry (IHC). IHC staining was performed using rabbit anti-rodent polyclonal antibodies for VEGF-A, VEGF-B, and VEGF receptors 1 and 2.

Results: IHC staining indicated varying intensity and location of VEGF-A, VEGF-B, VEGF-R1 and VEGF-R2 in ileum sections.  RLXN feed treatments diminished the histologic severity of NEC. VEGF factors increased in staining intensity and both VEGF receptors decreased in staining intensity in response to the RLXN treatments, but VEGF-B and VEGF-R1 had the most appreciable responses.

Conclusion: RLXN supplementation increases VEGF-B production while decreasing the histologic severity of NEC. Thus some of the therapeutic benefits of RLXN supplementation might be due to VEGF-B mediated maintenance of intestinal microvasculature. This interaction may provide further mechanistic direction for therapeutic interventions in NEC.

 

5.19 Oral Relaxin Alters Platelet-Derived Growth Factor Expression in NEC in Rats

Posted on December 22, 2014

E. D. Stamper1, P. Matheson2,3, A. Clarke1, J. Smith2, R. N. Garrison2,3, C. D. Downard4  1University Of Louisville,School Of Medicine,Louisville, KY, USA 2University Of Louisville,Department Of Surgery,Louisville, KY, USA 3Robley Rex Veterans Affairs Medical Center,Surgery,Louisville, KY, USA 4University Of Louisville,Pediatric Surgery,Louisville, KY, USA

Introduction:  Relaxin (RLXN) supplementation in experimental necrotizing enterocolitis (NEC) decreases severity of disease by mechanisms that include increased ileal blood flow via microvascular vasodilation.  However, RLXN also stimulates angiogenesis in adults, at least partially by up-regulating the platelet-derived growth factor (PDGF) family and analogous receptors.  Thus, we hypothesized that oral RLXN given with diet gavage might alter PDGF and PDGF receptor expression in experimental NEC in rats as compared to Controls or NEC rats alone.  

Methods:  We induced NEC in Sprague-Dawley rats by premature delivery, formula feeds, oral LPS, and episodic hypoxia/hypothermia.  NEC groups (n=12/group), randomized by litter, were: 1) NEC only, 2) NEC + 1x oral RLXN supplement (at 44 hours), and 3) NEC + oral RLXN supplement in All Feeds.  Controls (n=12) were vaginally delivered, dam fed, and time-matched.  At 48 hours, collected ileum sections were mounted in paraffin, and stained with either H&E or immunohistochemistry (IHC). IHC staining was performed using rabbit anti-rodent polyclonal antibodies for PDGF-A, PDGF-B, PDGF-Rα , or PDGF-Rβ .

Results: Table 1 describes the intensity of IHC staining in mucosal epithelial cells and severity of NEC in the ileum.  Briefly, PDGF-A, B, Rα , and Rβ  were present in Controls.  PDGF-B and PDGF-Rα  staining was increased in NEC, while PDGF-A and PDGF-Rβ  were decreased.  RLXN supplementation in the diet gavage in NEC rats increased PDGF-B expression and PDGF-Rα  was maintained.

Conclusion: RLXN supplementation increased PDGF-B expression, which is the primary isoform of the PDGF family involved in angiogenesis, and PDGF-Rα , while decreasing the severity of NEC.  These data suggest that the RLXN-mediated increase in angiogenic PDGF might play a role in the improvement of NEC and provide therapeutic direction for future studies with relaxin supplementation in NEC.

 

5.20 Quantitative Characterisation And Neurochemical Coding Of The Normal Human Hindgut Myenteric Plexus

Posted on December 22, 2014

K. S. Ng1,2, D. Mahns3, M. A. Gladman1,2  1Sydney Medical School – Concord, University Of Sydney,Academic Colorectal Unit,Sydney, NSW, Australia 2ANZAC Research Institute, University Of Sydney,Enteric Neuroscience & Gastrointestinal Research Group,Sydney, NSW, Australia 3University Of Western Sydney,Department Of Integrative Physiology, School Of Medicine,Sydney, NSW, Australia

Introduction:
Recent appreciation of enteropathies characterised by impaired gut motility secondary to aberrations of intrinsic nerve structure and/or function (GI neuromuscular disorders) has only served to highlight gross inadequacies in our understanding of the human enteric nervous system (ENS). Indeed, most of our current knowledge is derived from animal studies that have used inaccurate techniques such as unpaired tissue sections rather than ‘gold-standard’ paired wholemount preparations. Therefore, this study aimed to quantitatively investigate and neurochemically code the myenteric plexus of the human hindgut using paired wholemount samples of colon and rectum.

Methods:
Paired samples of human colon and rectum were procured from anterior resection specimens. The tissues were pinned flat in both relaxed and stretched states and fixed in Zamboni’s fixative. Wholemounts of colonic and rectal myenteric plexi were prepared by dissecting the mucosa, submucosa, and circular muscle off the longitudinal muscle. Myenteric neuronal immunostaining was performed using anti-Hu, anti-NOS, and anti-ChAT primary antibodies. These antibodies were secondarily labelled with fluorescent dyes. Wholemount images (approximately 100mm2) were acquired using an epifluorescence microscope equipped with a motorised stage to allow accurate assessment of ganglionic density, average ganglionic size, ganglionic area density, and neuronal density. ‘Stretch-corrected’ values were obtained taking account of tissue dimensions in the stretched and relaxed state. Data from paired colonic and rectal tissues were compared using the Wilcoxon signed-rank test.

Results:
Overall, 12 paired samples of colon and rectum were studied, all of which produced high quality immunostains permitting detailed analysis. Ganglia and individual neurons were readily identified and counted, and the fluorescent dyes discriminated according to their spectral wavelengths. Stretch-corrected ganglionic densities were similar between colon and rectum (colon: median 564 ganglia/100mm2 [range 386–921], rectum: 581 [360–923]; P=0.70), as were average ganglionic sizes (colon: 57,047μm2 [42,350–90,363], rectum: 52,021 [38,701–90,210], P=0.43). Whilst ganglionic area density tended to be lower in the rectum (colon: 11.96 mm2 per 100mm2 [7.53–18.64], rectum: 9.76 [5.80–17.19], P=0.12), there was no overall difference in stretch-corrected neuronal densities (colon: 176.3 neurons/mm2 [107.4–357.3], rectum: 174.7/mm2 [94.7–313.3], P=0.58).

Conclusion:
This is the first study to use paired samples of human gut tissue and apply wholemount immunostaining techniques whilst accounting for tissue stretch to quantitatively assess and neurochemically code the myenteric plexus of the human hindgut. This has allowed the development of the first robust normative data set to advance our current understanding of the intrinsic innervation of the human gut.

5.02 Stem Cell Subpopulation Depletion in Bariatric Patients: A Novel Cause of Higher Morbidity/Mortality

Posted on December 22, 2014

M. W. Findlay1,2, M. Sorkin1, R. Rennert1, M. Januszyk1, P. Than1, M. Rodrigues1, Z. Maan1, A. Whittam1, D. Duscher1, H. Rivas1, H. P. Lorenz1, J. M. Morton1, G. Gurtner1  1Stanford University,Department Of Surgery,Palo Alto, CA, USA 2University Of Melbourne,Department Of Surgery Royal Melbourne Hospital,Parkville, VIC, Australia

Introduction: Adipose tissue is a rich source of human stem cells (hASC’s) that can support fundamental tissue processes such as neovascularization, tissue regeneration and immune modulation.  Despite increases in obesity, bariatric surgery and post-bariatric plastic surgery in developed countries, little is known about the impact of morbid obesity on adipose-derived stem cell populations and stem cell-mediated health. Bariatric surgery helps reduce obesity-related morbidity and mortality through multimodal effects on blood pressure, blood lipids and glucose homeostasis, but despite this, the risk profile for post-bariatric patients does not return to normal.  We examined stem cell populations in bariatric patients to determine whether morbid obesity could adversely impact hASC’s and stem-cell related health.

Methods: Under appropriate Institutional Ethics and patient consent, human adipose tissue samples were harvested during elective laparoscopic and aesthetic surgeries at Stanford University Medical Centre.  The stromal vascular fraction (SVF) was isolated, lineage depleted by Magnetic Assisted Cell Sorting (MACS) before the resulting cell suspension was sorted by Fluorescence-Activated Cell Sorting (FACS) using CD45, CD31 and CD34 labeled fluorophores.   A single-cell microfluidics approach was then applied to quantify gene expression in 96 genes across a spectrum of stemness, surface marker, neovascularization, replication, differentiation and common second messenger pathways. An algorithm was then applied to assess for clustering into specific subpopulations of stem cells with correlation between the groups.  Murine studies were undertaken in parallel to examine potential mechanisms for any effect.

Results:15 bariatric patients and 5 controls were enrolled in the study.  A nine-fold reduction in a specific subpopulation of mesenchymal stromal cells was identified in bariatric patients when compared with non-obese controls (See Figure).  This deficient subpopulation was maintained on dual and multi-channel clustering, indicating the significance of the subpopulation. Murine studies demonstrated a linkage with diabetes mellitus by displaying the same subpopulation deficiency in streptozotocin-induced diabetic animals. 

Conclusion:The clinical profile of morbid obesity is more complex than its effects on blood glucose, lipids and blood pressure.  We have demonstrated a specific subpopulation of stem cells that it deficient in obese individuals with potential relevance for stem cell-mediated processes such as wound healing, tissue repair and regeneration after ischemia.

 

4.14 Examining Chenodeoxycholic Acid Analogs as a treatment for C. difficile with an Agent-Based Model

Posted on December 22, 2014

D. A. Lyubashevsky2, G. An1  1University Of Chicago,Surgery,Chicago, IL, USA 2Washington University,School Of Engineering And Applied Science,St. Louis, MO, USA

Introduction:  A contributing factor to the development of Clostridium difficile infection is the effect of enteric bile acid composition on C. difficle spore germination. Taurocholic acid (TCA) promotes spore germination, while deoxycholic acide (DCA) and chenodoxycholic acid (CDCA) inhibit germination. The commensal flora converts TCA to DCA, thereby acting as an environmental control suppressing CDI. Alternatively, CDCA acts as a competitive inhibitor of TCA by binding the C. diff receptor that triggers germination, In a healthy gut microbiome, CDCA is metabolized by the healthy microbiome into lithocholate, which also inhibits germination. However, CDCA is much more rapidly absorbed by the gut epithelium than TCA, leading to a net decrease in CDI inhibitor capacity in an antibiotic-induced commensal-depleted gut. Analogs to CDCA demonstrate less metabolic conversion by commensals and intestinal absorption, and therefore may show greater resistance to alterations in the microbiome following systemic antibiotics. We use an Agent Based Model (ABM) to simulate the dynamics of CDI, the inhibitory nature of CDCA, and the role of CDCA-analogs as a possible therapeutic for CDI.

Methods:  We expanded upon a previously developed ABM of CDI (CDIABM) by adding in the effects of CDCA. These effects included the inhibitory effect of CDCA on germination of C. diff spores, and the secretory/absorptive epithelial dynamics of CDCA. Simulation experiments were performed to reproduce the generation of CDI, and its subsequent treatment with both anti-CDI antibiotics and fecal microbial transplant (FMT). Further simulation experiments were performed to examine the effect of different regimens of CDCA analogs not subject to metabolism by commensal microbes. 

Results: Simulation experiments successfully recalibrated the CDIABM to the addition of CDCA by reproducing known dynamics of the development of CDI and its response to anti-CDI antibiotics and FMT.  Simulations employing CDCA-analogs demonstrated a reduction in the bimodal induction of CDI, stabilizing the anti-germination potential of the bile acid composition by reducing the impact of CDCA fluctuations due to alterations in its metabolism by commensal flora and absorption by intact epithelial cells. Continued administration of CDCA-analogs led to reduced recurrence of CDI, though with a higher residual spore count.

Conclusion: The expanded CDIABM successfully incorporated an additional bile acid control mechanism involved in the pathogenesis of CDI, demonstrating the advantageous modular nature of agent-based models. The simulation of the prophylactic effect of CDCA analogs suggests a potential therapeutic role for these compounds, particularly as an adjunct to other therapeutic measures with the goal of reducing recurrent CDI.  We suggest the use of dynamic computational models such as the CDIABM can serve as a useful adjunct in the investigations of host-pathogen interactions in clinically relevant scenarios.

 

4.15 Profiling of Circulating Exosomal MicroRNAs in Neonatal Necrotizing Enterocolitis

Posted on December 22, 2014

Y. Zhou1, G. E. Besner1  1Nationwide Children’s Hospital,Department Of Pediatric Surgery,Columbus, OH, USA

Introduction: Necrotizing enterocolitis (NEC) is the leading cause of death in premature babies. The early diagnosis and differentiation of NEC from neonatal sepsis and of medical NEC from surgical NEC is critical, but has been challenging. Little progress has been made in discovering novel diagnostic and prognostic biomarkers for NEC. Exosomes shed by producer cells and released into bodily fluids (e.g. blood, urine), represent an active process of cell-to-cell communication within the body. They contain a complex mixture of microRNAs, messenger RNAs and proteins from the cell of origin, making them an ideal source for biomarker discovery and diagnostic development. Our goal was to profile the microRNA content of serum exosomes from patients with NEC in an attempt to distinguish them from patients with sepsis, and to distinguish medical from surgical NEC.

Methods: Gestational age and post-conceptual age-matched premature babies were divided into four groups [prematures without acute disease, non-NEC sepsis, medial-NEC (patients who recovered without surgery), and surgical-NEC (patients requiring surgery)]. 400 μl of pooled serum (4 patients/group; 100 μl/patient) was obtained from patients upon the initial development of symptoms. Serum exosomes were isolated and microRNA profiling performed on the circulating exosomes using a Human miRnome miR PCR Array. Differentially expressed microRNAs were confirmed and/or further evaluated by qRT-PCR of exosomal RNA from the same individuals, and from three additional different individuals with the same diagnosis.

Results: Isolated exosomes from patient serum were bi-membrane vesicles, 30-200 nm in diameter, and positive for the exosome markers CD63 and flotillin-1. Microarray analysis revealed significant alterations in the expression of hundreds of microRNAs that had expression levels up- or down-regulated more than two-fold. We found that patients with NEC had significant up-regulation of miR-106, miR-1245a, and miR-224, and down-regulation of miR-145, miR-192, Let-7a, and miR-146a, consistent with previous reports in patients with intestinal ischemia or inflammation. In addition, miR-106 and Let-7a are known to target mRNAs that encode the components of inflammatory or anti-inflammatory signaling pathways including nuclear factor-kappaB (NF-κB) and Interleukin-10. Furthermore, exosomal microRNAs that have not yet been reported as being altered during NEC emerged as potentially novel disease markers, including up-regulation of miR-1323 and miR-524 and down-regulation of miR-215 and miR-19a.

Conclusion: Dynamic changes occur in the microRNA content of circulating exosomes from NEC patients. Serum exosome profiling may identify discriminating microRNA signatures distinguishing non-NEC sepsis from medical-NEC, and for risk stratification for NEC progression and severity. Identification of a panel of microRNAs in circulating exosomes may allow the discovery of biomarkers that signal NEC development.

4.16 Early Targeted Antibiotic Therapy Decreases Experimental Necrotizing Enterocolitis

Posted on December 22, 2014

J. C. Lim1, B. Bell1, G. Jang1, D. Hawkins1, D. Thomas1, S. Papillon1, J. Golden1, J. Wang1, L. Wang2, A. Grishin1, H. R. Ford1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Pathology,Los Angeles, CA, USA

Introduction:

            Necrotizing enterocolitis (NEC) is the most common gastrointestinal surgical emergency among neonates.  The precise etiology of NEC is unknown, but risk factors include a susceptible host, enteral feeding, and bacterial colonization.  Opportunistic pathogens, such as Cronobacter muytjensii, have been identified in clinical outbreaks and confirmed as disease contributors in experimental models.  Current clinical management includes broad-spectrum antibiotics with variable results.  We hypothesized that antibiotic prophylaxis targeting opportunistic pathogens would decrease the incidence and severity of NEC.

 

Methods:

            NEC was induced in a neonatal rat model of thrice[CG1]  daily formula feeding and hypoxia.  The pups were separated into six treatment groups based on formula composition: baseline, early ampicillin (starting day of life #1), late ampicillin (starting day of life #3), C. muytjensii (every feed), early ampicillin with C. muytjensii, and late ampicillin with C. muytjensii.  Animals were sacrified on day of life #4.  The terminal ileum was histologically scored by H&E stain with scores ≥2 indicative of NEC.  The microbiota of the terminal ileum and daily stools were characterized by culture-based 16S rRNA sequencing.

 

Results:

            The baseline group produced an NEC incidence of 29%.  The opportunistic pathogen C. muytjensii increased incidence to 69% (p=0.0013 compared to baseline).  When early ampicillin was given in the presence of C. muytjensii, NEC incidence decreased to 25%, resembling baseline (p=0.9060 compared to baseline, p=0.0185 to C. muytjensii).  In contrast, when late ampicillin was given to rats exposed to C. muytjensii, the incidence remained high at 71% (p=0.0047 compared to baseline, p=0.7701 to C. muyjtensii).  Ampicillin alone, regardless of timing, increased NEC: early with 67% incidence and late with 75% incidence.

            Microbiota profiling revealed an overall paucity of bacteria in animals with NEC compared to those without NEC.  Utilizing the Shannon Index of diversity, no significant trends were found between treatment groups or NEC scores.

 

Conclusion:

            Targeted antibiotic therapy was only effective in the presence of the opportunistic pathogen and only if started early.  In the absence of the opportunistic pathogen, the antibiotic treatment was harmful rather than beneficial.  Our findings suggest that neonates at risk for NEC should undergo routine surveillance for opportunistic pathogens in their stool followed by targeted antibiotic therapy for these isolates.  Further studies are indicated to investigate the similarities between the opportunistic pathogens and empiric antibiotic groups.

4.17 Luminal Benzalkonium Chloride: A Non-Invasive Model of Functional Bowel Obstruction

Posted on December 22, 2014

W. N. El-Nachef1, M. K. Collins1, T. C. Grikscheit1,2  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2University Of Southern California,Keck School Of Medicine,Los Angeles, CA, USA

Introduction:
Functional bowel disorders such as colonic pseudo-obstruction, idiopathic megacolon, Hirschsprung's disease, and postsurgical ileus are incompletely understood. Models of functional bowel obstruction will assist in exploring the pathophysiology of these entities; however, previous models rely on mechanical means to obstruct bowel. Benzalkonium chloride (BAC) was first shown in the 1970s to ablate the myenteric plexus in rats when applied to the serosal surface of bowel. However, we have found this approach to be limited by the need to perform laparotomy, the wide spread and difficult to control field of exposure, and the difficulty to reproduce nonlethal obstruction in mice.  Here, we describe a non-invasive protocol in which BAC is applied to the luminal surface of murine colon, leading to ablation of the submucosal plexus and a dramatic functional obstruction.

Methods:

Male C57/BL6 mice aged 2 months were selected to receive PBS, 5% BAC, or 10% BAC per rectum, with 3 mice per group. Extra-small craft cotton swabs were soaked in the respective solution and then inserted per rectum until the bottom-most part of the cotton applicator was just beyond the anus and then left in place for 4 minutes. The cotton swab was then removed with gentle counter-pressure to prevent prolapse.  This was repeated for a total of 4 treatments, with the last cotton swab duration being 3 minutes, for a total of 15 minutes of exposure.

The mice were observed daily and sacrificed on post-exposure day 5. Colons were resected and prepared into paraffin blocks for histologic analysis with hematoxylin and eosin (H&E) and immunofluorescent (IF) staining with antibodies to the pan-neuronal marker TUJ-1 and smooth muscle actin.

Results:
At time of resection, all PBS-treated colons appeared phenotypically normal, with discrete fecal pellets visible within the lumen. Colons treated with 10% BAC were uniformly and massively obstructed with rigid-dilatation due to impacted feces; H&E revealed an intact epithelium but an enlarged submucosa  with cellular infiltrate, and IF staining revealed an absence of TUJ-1 in the submucosa but sparing of the myenteric plexus. Colons treated with 5% BAC had less severe dilatation, a smaller submucosal infiltrate, and scant TUJ-1 staining in the submucosa. PBS-treated mice displayed normal histology and IF staining patterns.

Conclusion:
 We have demonstrated that BAC can be applied to the luminal surface of intestine to effect a functional obstruction, the severity of which appears to be dose dependent. This obstructive phenotype is accompanied by the decreased/absent visualization of submucosal neurons on IF staining, though myenteric neurons appear intact. This suggests that perturbations to the submucosal plexus alone can result in functional obstructive disorders. This protocol is non-invasive, not dependent on mechanical obstruction, and technically simple; thus, it can be easily replicated to model functional bowel disorders.  

4.18 THROMBELASTOGRAPHY PERFOMRED WITHOUT AN ACTIVATOR ENHANCES DETECTION OF FIBRINOLYSIS

Posted on December 22, 2014

B. A. Quinn1,2, E. Gonzalez1, H. B. Moore1, M. P. Chapman1, A. Sauaia1, A. Banerjee1, C. C. Silliman1,3, E. E. Moore1,2  1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2Denver Health Medical Center,Department Of Surgery,Denver, CO, USA 3Bonfils Blood Center,Research Department,Denver, CO, USA

Introduction: The fibrinolytic response to trauma can be physiologic (preventing systemic clot propagation), pathologic (favoring bleeding), or shut-down (favoring un-regulated clotting). Thrombelastography (TEG) is used to quantify fibrinolysis and facilitate appropriate use of fibrinolysis inhibitors such as tranexamic acid in bleeding patients with hyperfibrinolysis. TEG in the trauma bay is typically performed with tissue factor as an activator (rapid-TEG). However, TEG can also be performed with kaolin as an activator or as a “native assay” with no activator. Which TEG modality is optimal for detecting fibrinolysis remains to be elucidated. We hypothesized that the use of kaolin or tissue factor as activators for TEG decreases the assay’s capability to detect tissue plasminogen activator (tPA)-induced fibrinolysis.

 

Methods: Citrated and non-citrated blood samples were collected from 10 healthy adults. Fibrinolysis was achieved by addition of tPA to whole blood samples prior to running the assay in five concentrations: 0, 50, 75, 150, and 300 (ng/mL). tPA-induced fibrinolysis was quantified by three different TEG methods—citrated native (CN) (no activator), citrated kaolin (CK), and non-citrated rapid (RT) (tissue factor). The TEG variable of fibrinolysis, LY30 (percent clot lysis at 30 minutes after reaching maximum clot strength), was compared at each tPA dose amongst CN, CK, and RT. The significance of differences between groups was tested by the Friedman’s test (p<0.05). Groups with significant differences were subjected to a post-hoc pairwise comparison with Bonferroni adjustment.




Results: At baseline (0 tPA), RT vs. CN detected a greater LY30 (2.8 vs. 1.7%, p=0.03). At 50 tPA the differences in LY30 among RT, CK, and CN were not statistically significant (p=0.050). At 75 tPA, CN vs. RT detected a greater LY30 (15.8 vs. 3.6%, p<0.001). At 150 tPA, CN vs. RT detected a greater LY30 (56.1 vs. 36.7%, p=0.005). At 300 tPA there were no significant differences.

Conclusion: Using a coagulation activator decreases the threshold for detecting tPA-induced fibrinolysis by TEG. When fibrinolysis was induced to levels of tPA previously reported in trauma patients (75 and 150 ng/mL), CN detected more fibrinolysis. Early detection of hyper-fibrinolysis in injured patients is imperative for triggering treatment with anti-fibrinolytics in order to control bleeding and decrease mortality. Our data demonstrates that TEG can be used with no activator (CN) for adequate quantification of fibrinolysis.

4.19 Cytokine Gene Expression in the Gastrocnemius of Patients with Peripheral Arterial Disease

Posted on December 22, 2014

L. A. Carpenter1, J. R. Thompson1, D. M. Ha1, S. A. Swanson1, J. M. Johanning1,2, E. A. Papoutsi1, P. Koutakis1, D. A. Miserlis1, I. I. Pipinos1,2, G. P. Casale1  1University Of Nebraska Medical Center,Department Of Surgery,Omaha, NE, USA 2VA Nebraska-Western Iowa Health Care System,Omaha, NE, USA

Introduction:
Peripheral arterial disease (PAD) is characterized by limb dysfunction in association with cycles of ischemia/reperfusion that cause oxidative damage to muscles of the lower leg.  Studies evaluating the contribution of inflammation to the pathology of PAD identified increased serum concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin 6 and monocyte chemoattractant protein-1.  Recently, we demonstrated increased concentrations of cytokines including TNF-alpha, transforming growth factor beta1 (TGF-beta1) and chemokine (c-c motif) ligand 5 (CCL5) in the gastrocnemius of PAD patients.  In this study, we tested the hypothesis that these increases were due to increased gene expression in the gastrocnemius.

Methods:
Gastrocnemius biopsies were collected from PAD patients (N=24) at Fontaine Stage II and controls (N=18) with no leg impairment.  Muscle homogenates were analyzed by qPCR for TNF-alpha, TGF-beta1 and CCL5 gene transcripts expressed as fold change in relation to myosin heavy chain transcripts.

Results:
TNF-alpha, TGF-beta1 and CCL5 transcripts were increased in PAD patients (mean +/- s.e.; 1.07 +/- 0.31, 12.87 +/- 3.38 and 3.01 +/- 0.99, respectively) compared to controls (0.02 +/- 0.01, 0.56 +/- 0.25 and 0.15 +/- 0.10) at p values of 0.003, 0.001 and 0.009.  Relative changes in these transcripts are in close agreement with changes in protein expression determined in our previous study.

Conclusion:
The data support increased, local expression of the genes for TNF-alpha, TGF-beta1 and CCL5 in the gastrocnemius of PAD patients and identify a local cytokine milieu that suppresses myoblast differentiation and promotes fibrotic replacement of oxidatively damaged and necrotic myofibers.

4.20 Acute Hind Limb Ischemia in the Recombinant Polygenic Type 2 Diabetic Mouse

Posted on December 22, 2014

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

Introduction: The polygenic mouse model of diabetes is believed to better simulate the human adult type-2 diabetes compared to the monogenic models (i.e. db/db or Ob/Ob). This model exhibits a maturity-onset transition from impaired glucose tolerance to a stable non-fasting hyperglycemia when fed a 10% high fat diet after 16 weeks. Wound healing experiments using these mice show substantial impairment in wound healing processes involving the skin. The aim of this study was to assess acute skeletal muscle injury in the polygenic mouse model of diabetes following hind limb ischemia reperfusion (IR).
 

Methods: The recombinant polygenic diabetic mice (NONcNZO10/LtJ, n=6) and its non-diabetic control strain (NON/ShiLtJ, n=5) were subjected to unilateral moderate hind limb tourniquet ischemia for 1.5 hours followed by 24 hours of reperfusion. To confirm their diabetic state, fasting blood glucose levels were measured prior to ischemia. After 24 hours of reperfusion, mice were sacrificed and muscle samples were processed for histological quantitative assessment of muscle fiber injury and inflammatory cell infiltration (Ly6G, marker of neutrophils, or Mac-3, marker of monocytes lineage) using immunohistochemistry. The protein levels of pro-inflammatory chemokine KC (CXCL1) in the serum and solubilized muscle protein extracts were measured using ELISA. Data were expressed as mean±SEM and statistical analysis was performed using student’s t-test.

Results: The fasting blood glucose levels in the diabetic mice were significantly greater than in the non-diabetic mice (472±32 vs. 165±28 mg/dL, p<0.000001). There was no significant difference in the degree of muscle fiber injury between the diabetic vs. non-diabetic mice (15±2 vs. 16±2 average injured fiber per high power field, p=0.6). The accumulation of Ly6G+ (41±10 vs. 48±15 average positive cells per field, p=0.7) and Mac3+ (42±6 vs. 33±5 average positive cells per field, p=0.7) cells in skeletal muscle following IR was similar in the diabetic vs. non-diabetic mice. Furthermore, levels of muscle KC (17±2 vs. 14±2 pg/mg protein, p=0.2) and serum KC (103±6 vs. 73±15 pg/ml, p=0.09) were also not statistically different between the two groups.

Conclusion: While excessive IR injury and increased inflammation is believed to play a major role in defective wound healing models, the pattern of acute skeletal muscle IR in the polygenic diabetic mouse does not appear to be worse than that of the non-diabetic mouse following 1.5 hours of ischemia. Further studies in these polygenic diabetic mice subjected to severe periods of ischemia (i.e. ≥3 hours) and characterization of the regenerative phase (i.e. healing) in the limb muscle is warranted.

4.01 Valproic Acid Alters Inflammatory Gene Expression after Traumatic Brain Injury and Hemorrhagic Shock

Posted on December 22, 2014

T. Bambakidis1, S. E. Dekker1, M. Sillesen1, B. Liu1, C. N. Johnson1, I. Halaweish1, Y. Li1, H. B. Alam1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction: We have reported that valproic acid (VPA) can create a pro-survival gene expression profile in various models of lethal insults, and its administration can significantly decrease brain lesion size and surrounding inflammation, in a swine model of combined traumatic brain injury (TBI) + hemorrhagic shock (HS). It, however, remains unknown whether this neuroprotective effect is driven by alterations in the expression of cerebral inflammatory genes.

Methods: Computer-controlled TBI (cortical impact) and HS (40% blood volume) were induced in 10 Yorkshire swine. After two hours of shock, animals were randomly treated with either 6% hextend (HEX; 1x shed blood) or HEX+VPA (300mg/kg) (n=5/group). Six hours after resuscitation, brains were harvested, RNA isolated, and gene expression profiles measured using a Porcine Gene ST 1.1 microarray (Affymetrix, CA). Ingenuity Pathway Analysis® (IPA), Gene Ontology (GO), and Parametric Gene Set Enrichment Analysis (PGSEA) were used for pathway analysis. Key microarray findings were verified using real-time polymerase chain reaction (PCR).

Results: Of the 1753 genes modulated by VPA, significant alterations were noted in genes related to the inflammatory response. IPA analysis revealed that VPA significantly down-regulated the complement system (P<0.001), natural killer cell communication (P<0.001), and dendritic cell maturation (P<0.001) (Figure). Real-time PCR data confirmed that VPA significantly decreased the expression of genes associated with inflammation, such as CCR1 (P=0.01), IL-1β (P=0.003), TREM2 (P=0.02), and TYROBP (P=0.05).

Conclusion: This is the first high-throughput analysis of cerebral gene expression profile following TBI+HS which reveals that VPA treatment significantly attenuates inflammatory pathways.

 

4.02 The Role of Erythropoietin and Hepcidin in the Regulation of Persistent Injury-Associated Anemia

Posted on December 22, 2014

I. G. Alamo1, K. B. Kannan1, M. A. Smith1, P. A. Efron1, A. M. Mohr1  1University Of Florida,Surgery,Gainesville, FL, USA

Introduction:  The cause of persistent injury-associated anemia is multifactorial and includes blood loss, impaired proliferation of erythroid progenitor cells, altered erythropoietin (EPO) response, dysregulation of iron homeostasis, and chronic inflammation/stress. Hepcidin plays a key role in iron homeostasis and has been shown to be regulated by anemia as well as inflammation and EPO is a main regulator of erythropoiesis induced by hypoxia. The relationship between these two factors in persistent injury-associated anemia has yet to be fully elucidated. Using a combined lung injury (LC)/hemorrhagic shock (HS)/chronic restraint stress (CS) model to produce persistent injury-associated anemia, the aim of this study was to investigate the regulation of hepcidin and EPO.

 

Methods: Male Sprague-Dawley rats (N=6-9 per group) were randomly assigned into one of the four groups of rodent models: naïve, CS alone, combined LCHS, or LCHS/CS.  CS was performed using restraining cylinders every day for two hours following either LC or LCHS. During CS, rodents were exposed to 80-85 decibel alarms for two minutes every 30 minutes and rotated to prevent habituation. At day seven, blood, urine, bone marrow and lung tissue was harvested. Hemoglobin (Hgb), EPO, and hepcidin levels were assessed.  Data presented as mean±SD in each group. *p<0.05 vs naive

 

Results: Compared to naïve rodents, plasma hepcidin levels were significantly decreased in CS, LCHS, and LCHS/CS groups (Figure).  Similarly, urine hepcidin levels were significantly lower in CS, LCHS and LCHS/CS as compared to naïve (12±4*, 10±3*, 10±2* vs. 52±26 pg/mg protein). There was no change in bone marrow hepcidin mRNA levels in any group.  In the LCHS/CS group, there was a significant 70% decrease in lung hepcidin mRNA level as compared to naive. Only LCHS/CS was associated with persistent anemia despite significant elevation of EPO (Figure).  There was a strong inverse correlation between EPO and plasma hepcidin (Pearson R= -0.362, p<0.05).

 

Conclusion: Chronic stress, LCHS and LCHS/CS all significantly decrease plasma and urine hepcidin. Yet only LCHS/CS is associated with persistent anemia despite elevation of EPO. Although there is an inverse correlation between hepcidin and EPO in this model, anemia alone does not regulate hepcidin. The addition of chronic stress did not counteract hepcidin suppression.  Further study of the mechanisms involved in injury-associated persistent anemia is warranted.

 

4.03 Daily Propranolol Prevents Prolonged HPC mobilization in a Chronic Stress and Polytrauma Model

Posted on December 22, 2014

L. E. Bible2, L. V. Pasupuleti2, A. V. Gore2, Z. C. Sifri2, A. M. Mohr1  1University Of Florida,General Surgery,Gainesville, FL, USA 2New Jersey Medical School,Newark, NJ, USA

Introduction:  Following injury, hematopoietic progenitor cells (HPC) mobilize to the peripheral blood from the bone marrow (BM) and then home to the injured tissue. We have previously shown that propranolol decreases HPC mobilization and improves BM function following acute injury in rodent models. These acute injury models do not reflect the prolonged period of critical illness following severe traumatic injury. Using our unique lung injury (LC)/hemorrhagic shock (HS)/chronic restraint stress (CRS) model, we hypothesize that daily propranolol administration following LC/CRS and LCHS/CRS will reduce prolonged HPC mobilization without worsening lung healing.

Methods:  Male Sprague-Dawley rats (n=5-9/group) underwent six days of CRS after undergoing LC or LCHS. CRS consists of a daily two hour period of restraint within a cylinder that is interrupted every 30 minutes by alarms and repositioning. Each day following their intervention, the rats received intraperitoneal propranolol (10mg/kg). On day seven the peripheral blood was analyzed for granulocyte-colony stimulating factor (G-CSF) via ELISA and for HPC mobilization using c-kit and CD71 flow cytometry, and the lungs were examined histologically to grade injury.

Results: As previously shown, seven days following LC and LCHS, the addition of CRS significantly increased HPC mobilization which is associated with persistently elevated G-CSF and worsened lung injury scores (Table). The addition of propranolol to LC/CRS and LCHS/CRS models significantly reduces HPC mobilization in peripheral blood (Table). In addition, the administration of propranolol following LC/CRS and LCHS/CRS restores G-CSF levels to that of naïve animals without worsening lung injury scores. 

Conclusion: Daily propranolol administration following both LC/CRS and LCHS/CRS reduces prolonged HPC mobilization from the bone marrow and decreases plasma G-CSF levels. Despite the reduction of HPC mobilization, the lung healing did not worsen. Alleviating chronic stress with propranolol may be a future therapeutic target to improve healing following severe injury.

 

4.04 Interleukin-6 is Essential for Endogenous Fibrinogen Release in the Acute Phase Response to Trauma

Posted on December 22, 2014

R. A. Jacobson1,2, J. G. Schoenecker1,3  1Vanderbilt University Medical Center,Pharmacology,Nashville, TN, USA 2Rush University Medical Center,Rush Medical College,Chicago, IL, USA 3Vanderbilt University Medical Center,Orthopedics,Nashville, TN, USA

Introduction: Fibrinogen (FBG) is an acute phase reactant secreted from the liver in response to injury and consumed during hemostasis at sites of tissue damage. Recent studies show that patients deficient in FBG due to traumatic consumption or dilution secondary to fluid resuscitation are coagulopathic, with an elevated risk of further bleeding. As such, repletion of FBG is a crucial step in the endogenous and therapeutic responses to injury. However, the mechanism of induction of FBG secretion after injury is incompletely understood. This study investigated the role of the inflammatory mediator interleukin-6 (IL-6) in endogenous FBG secretion following blunt trauma. Our hypothesis is that trauma-induced circulating IL-6 is essential for upregulation of circulating FBG.

Methods: 8-week-old male wild type C57BL/6 (WT) and IL-6 deficient (IL-6-/-) mice were injured, then sacrificed at the time points indicated below (n=3 mice per time point) by CO2 inhalation with cardiac puncture for blood collection. Citrated blood was processed into plasma, and ELISA for FBG and IL-6 were performed.  Injury was induced using a modified version of the method described by Pape et al (J Surg Res 2011). Open muscle trauma was induced by clamping a large needle driver around the body of the gastrocnemius for 30s.

Results: Figure 1A shows plasma levels of IL-6 before and after injury in WT mice. IL-6 peaks above 60pg/mL 4-8 hours post-injury from a baseline (t=0) below 5pg/mL.  Levels return to baseline at 48 hours post-injury. Plasma IL-6 was undetectable in IL-6-/- mice.

Figure 1B shows plasma fibrinogen levels in WT and IL-6-/- mice following injury. In WT mice, FBG levels began to rise at the earliest time point – 4 hours, peak at 24 hours and then decline. In IL-6-/- mice, there is no immediate rise in FBG in response to trauma. A blunted, delayed increase in circulating FBG does occur between 24 and 48 hours post-injury.

Conclusion: The results presented in this study indicate that circulating IL-6 is essential for physiologic upregulation of FBG in response to trauma. This finding is in line with past work showing that IL-6 is regulates hepatic secretion of additional coagulation proteins.  In this sense, IL-6 can be viewed as an “SOS” signal released from damaged tissue, inducing the production of essential hemostatic proteins consumed at the site of injury. This schema is illustrated in Figure 1C. Future studies are needed to determine the mechanism of IL-6 release from damaged tissue, and its induction of FBG secretion from the liver. These studies could serve as proof of principle for therapeutic trials designed to treat the pathophysiologic conditions of diminished (hypocoagulable) or excessive (hypercoagulable) circulating FBG.

4.06 An Accurate Method For Predicting Death From Sepsis

Posted on December 22, 2014

J. W. Kuethe1, E. F. Midura1, K. R. Kasten2, C. M. Freeman1, T. C. Rice1, C. C. Caldwell1  1University Of Cincinnati,Division Of Research,Cincinnati, OH, USA 2East Carolina University Brody School Of Medicine,Department Of Surgery,Greenville, NC, USA

Introduction: The successful early immune response to sepsis strikes a balance between microbial eradication and host tissue injury. Unsuccessful clearance often results in persistent inflammatory / immunosuppressive catabolic syndrome (PICS).  Due to a fluctuating inflammatory state, a measure of immune status and an accurate model of risk stratification are critical to the effective use of immune modulating therapies. Determination of leukocyte numbers, their activation state, and cytokine levels has been proposed to stratify such patients. In mice, circulating IL-6 levels allow for risk stratification following cecal ligation and puncture (CLP). Although CLP is the gold standard for inducing sepsis in experimental murine models, lack of source control is a severe limitation when extrapolating to sepsis management in humans. Therefore, we utilized a CLP-Excision (CLP-E) model in which cecal excision, peritoneal wash and antibiotic treatment were performed following CLP. Using this more clinically relevant model, we hypothesized that leukocyte characterization and cytokine measurements, isolated at the time of source control, would allow us to predict survival.

Methods: Outbred mice were subjected to CLP (50% ligation / 20 gauge puncture). After CLP, the their abdomens were re-explored, the necrotic cecums debrided, the abdomens washed and a single intra-peritoneal dose of antibiotics administered. Survival was then monitored. The peritoneal wash was analyzed for IL-6 concentration by ELISA, and neutrophil numbers and activation by flow cytometry.

Results: Following excision, neutrophil characteristics and wash IL-6 levels were analyzed. After assessing for survival, the measurements associated with the mice that lived (LIVE) and those that died (DIE) were used to generate an ROC curve. Two ROC curves were significant in predicting survival (Table).  A marker of neutrophil activation, CD11b was noted to be 67% more elevated in the LIVE group compared to the DIE group (p<0.0001). IL-6 concentration, a marker of inflammation, was observed to be increased 2.2 fold in the DIE group compared to the LIVE group (p<0.001). In a subsequent cohort, neutrophil CD11b and IL-6 accurately predicted risk of death using the appropriate ROC curve.

Conclusion: This technique predicts survival by analyzing surgical waste in a clinically relevant model.  We observed that neutrophil activation was blunted in the DIE cohort, but elevated in the LIVE cohort. Based on this observation, we speculate that treatments to increase neutrophil activation in the DIE cohort would improve survival, but would only exacerbate host tissue injury in the LIVE cohort, thus demonstrating a need to determine immune status prior to considering immune modulating therapies.

 

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