63.01 Screening of Novel Synthesized Analoges Targeting Histone Deacetylase in Aggressive Thyroid Cancers

Posted on December 22, 2014

S. Jang1, X. Yu1, S. K. Odorico1, M. Clark1, C. Schienebeck2, W. Tang2, H. Chen1  1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Chemistry,Madison, WI, USA

Introduction: There are limited effective therapies for aggressive thyroid malignancies including anaplastic and poorly differentiated cancers. Compounds targeting histone deacetylases (HDAC) have shown promising antitumor activities in others cancers.  In order to develop novel therapies for these aggressive thyroid cancers, we synthesized a new group of analogues targeting HDACs named AB1 to AB13 which has different linkers between a metal chelating group and a hydrophobic cap. Therefore, the purpose of this study was to screen out the most effective compounds and evaluate the therapeutic efficacy in aggressive thyroid cancers.

Methods: Anaplastic (HTh7 and 8505C) and metastatic follicular (FTC236) thyroid cancer cells were treated with thirteen AB analogues using various concentrations, and the IC50 was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.  The most effective compounds were selected based on the IC50 to further study the molecular mechanisms of growth regulation. Both cell cycle regulatory proteins and apoptosis markers were analyzed by the Western blot. In addition, the expression levels of the thyrocyte-specific genes were quantified by real-time PCR to assess the drug potency of inducing re-differentiation in aggressive thyroid cancer cells.

Results: Among all the analogues, AB1, 4, 5, 6, 13 showed very limited cytotoxicity effect while AB7, 8, 9, 11, and 12 presented with moderate efficacy to inhibit cell growth. AB2, AB3, and AB10 demonstrated the lowest IC50 values of the thirteen screened drug analogues (Table 1). The AB analogues showed less cytotoxicity against human fibroblasts WI38. AB2, AB3, and AB10 treatment resulted in an increase of apoptosis markers including cleaved poly adenosine diphosphate ribose polymerase (PARP) and cleaved caspase 3 in a dose dependent manner in all three cancer cell lines. Additionally, the expression of cell cycle regulatory proteins p21/WAF1 and p27 Kip increased with the treatment of ABs while cyclin D1 decreased dose-dependently. Furthermore, AB2, AB3, and AB10 were able to induce various of thyrocyte specific genes in all the cell lines indicated by increased level of sodium iodide symporter (NIS), paired box gene 8 (PAX8), thyroid transcription factor 1 (TTF1), TTF2, and thyroid stimulating hormone receptors (TSHR).

Conclusion: Novel synthetic HDAC inhibitors AB2, AB3 and AB10 suppress thyroid cancer cell growth via cell cycle arrest and apoptosis. They also induce cell re-differentiation which could make aggressive thyroid cancer cells more susceptible to radioactive iodine therapy. Therefore, these compounds could be new options for patients with aggressive thyroid cancers.

 

63.02 A Dynamic Model of In-Vitro Thrombogenicity Testing for Heart Valves

Posted on December 22, 2014

M. R. Helder1, B. Tefft2, R. Hennessy2, C. D. Koch4, D. B. Spoon2, R. D. Simari3, A. Lerman2  1Mayo Clinic,Cardiovascular Surgery,Rochester, MN, USA 2Mayo Clinic,Cardiovascular Diseases,Rochester, MN, USA 3University Of Kansas,School Of Medicine,Kansas City, KS, USA 4Mayo Clinic,Clinical Core Laboratory Services,Rochester, MN, USA

Introduction:   One goal of tissue engineering is to develop heart valves for implantation with reduced thrombogenicity.  Current in-vitro thrombogenicity tests are static and do not reflect the true thrombogenicity potential of valves.  We present a novel in-vitro method to dynamically test the thrombogenic potential of heart valves and present our experiments for optimizing test conditions. 

Methods:   The Haemobile (Haemoscan, Inc) was modified for in-vitro thrombogenicity testing of heart valves with intact roots.  The Haemobile is comprised of a polycarbonate fluid circuit that contains the valve, which is mounted onto a motor that creates pulsatile fluid flow without a pump system.  Mechanical valves with aortic conduits were utilized in this study to validate the model and establish a positive control as well as optimal conditions for future studies.  Various conditions were tested including temperature (4ºC, 25ºC, 37ºC), run time (3 hr, 5 hr), and blood:saline dilution (100% blood, 50% blood, 40% blood, 35% blood, 30% blood).  Blood was obtained in an aseptic manner from sheep and immediately used for testing.  Kaolin activated thromboelastography (TEG® 5000, Haemonetics,) was performed in duplicate to measure the clotting potential of blood samples collected prior to valve exposure and at specified time points following valve exposure. Thromboelastography showed clot potential at every dilution prior to starting the run.  Any clots produced in the circuit were weighed.

Results:  The optimal temperature for testing was determined by exposing undiluted blood to a mechanical valve for 5 hr. A macroscopic clot was produced only at 25ºC, and thus, this temperature was used in subsequent experiments.  The shortest test time that formed a thrombus was 3 hr and this was the time point utilized in further studies.  Blood dilutions were tested in order to create more stringent criteria to differentiate valve thrombogenicity.  Dilutions of ≥ 40% blood created a clot that decreased linearly by mass with increasing dilution of blood samples.  Correspondingly, TEG readings of the blood sample were more depressed with larger clots indicating that increasing amounts of coagulation factors and platelets were utilized (Figure). 

Conclusions:  The Haemobile provides a dynamic method to test the thrombogenicity of heart valves.  A clot formed on a mechanical valve with dilutions ≥ 40% blood following 3 hr of exposure at 25ºC.  In this model, there was excellent agreement between blood sample dilution, mass of thrombus and TEG readings allowing for small differences in valve thrombogenicity to be detected.  Future studies will test the thrombogenicity of decellularized and other tissue engineered heart valves.

63.03 Mechanisms of Erythropoietin-Mediated Neuroprotection Following Spinal Cord Ischemia Reperfusion

Posted on December 22, 2014

L. S. Foley1, J. Mares1, F. Puskas1, M. T. Bell1, D. T. Bennett1, K. Freeman1, M. Weyant1, D. A. Fullerton1, T. B. Reece1  1University Of Colorado Denver,Surgery/Cardiothoracic,Aurora, CO, USA

Introduction:  Delayed paraplegia is a devastating complication of thoracoabdominal aortic surgery.  Erythropoietin (EPO) attenuates this injury in models of spinal cord ischemia. Proposed mechanisms include induction of ischemic tolerance in normal tissue and activation of an ischemia-induced receptor, which attenuates neuronal loss in ischemic tissue. This study aims to elucidate mechanisms of EPO-mediated neuroprotection.  We hypothesized that EPO acts on ischemic tissue to limit neuronal loss.  

Methods:  Adult male C57/BL6 mice underwent sternotomy and 4-minute thoracic aortic crossclamp. Seven pretreatment mice received EPO (20U/kg) via intraperitoneal injection 24 hours prior to operation. Seven perioperative mice received the same EPO dose 4 hours prior to operation. Seven controls received 0.9% saline.  Four sham mice underwent saline administration and sternotomy without crossclamp. Functional outcomes were measured using Basso motor score for 48 hours (Basso score ranges 1-9; 1 refers to complete paralysis and 9 is normal hind-limb motor function).  Spinal cords were harvested and homogenized for biochemical analysis. 

Results:  Ischemia reperfusion (IR) injury uniformly induced paraplegia in control mice (p<0.01).  Synchronous EPO administration with IR prevented motor decline, which occurred in ischemic controls and EPO pretreated mice starting at 36 hours post-op (mean Basso 6.43 vs 1.86 and 3, respectively; p<0.01).  All ischemic control and pretreatment mice progressed to paraplegia, with no significant difference in hind limb function at all time points.

Conclusion:  EPO administration attenuates functional decline following spinal cord ischemia reperfusion injury when administered concomitant to the injury, but this effect is lost when EPO is given as a pretreatment.  These findings point towards a potential salvaging role for EPO following neurologic injury. Elucidation of mechanisms involved in spinal cord protection is essential for reducing delayed paraplegia. 

 

63.04 Assessment of Swab and Biopsy Sampling Methods for Vocal Fold Microbiota Studies

Posted on December 22, 2014

S. Tadayon1, A. Hanshew1, S. Thibeault1  1University Of Wisconsin,Madison, WI, USA

Introduction:
The microbiota is comprised of bacteria, fungi, and archaea that reside on epithelial surfaces throughout the body. Shifts in normal microbial communities have now been linked to disease in numerous body sites. We sought to compare two sampling methods for microbiota study in healthy vocal folds, including swabbing and biopsies. Biopsy of true vocal folds is problematic because it can lead to scarring and dysphonia and therefore cannot ethically be used to evaluate healthy vocal folds in humans. Swabbing has been used to assess microbial communities in other areas of the body because it is less invasive and does not disrupt the normal epithelium. If swabbing of vocal folds reveals similar microbial communities to samples taken by biopsy, swabbing could be used as an alternative to biopsy. Additionally, the microbial communities of false vocal folds could be used in clinical practice as proxies for true vocal folds in research, but the similarity between these two niches is unknown. The false and true vocal folds differ in epithelial type and in function. 

Methods:

Due to their use as an animal model in vocal fold research, we used 6 pigs in our comparison of the microbial communities in vocal folds. True and false vocal fold mucosal samples were collected using sterile Catch-All swabs and biopsy. Total DNA was extracted and the V3-V5 region of the 16S rRNA gene was amplified.  The resulting amplicons were sequenced using 454 pyrosequencing.

Results:
Preliminary analysis of data from three pigs suggest that microbial communities are unique in relative composition for each pig, though all three communities were dominated by bacteria in the phyla Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria. The microbial communities found by swabbing were similar to those found by biopsy. We did not find a relationship between the microbiota of true and false vocal folds in the preliminary data from the first three pigs. 

Conclusion:

The microbiota of swabbed and biopsied samples were similar suggesting that swabbing could be used as a reliable alternative to biopsy. Swabbing will allow for greater assessment of microbial communities in healthy individuals since it is much less traumatic to the epithelium and does not cause scarring. 

 

63.05 Nanofiber Mesh Scaffolding for Recreation of the Abdominal Wall in a Laboratory Rat

Posted on December 22, 2014

R. Restrepo1,2, B. Hoagland1, J. Stephenson1,2  1Naval Medical Center Portsmouth,Portsmouth, VA, USA 2Uniformed Services University,Bethesda, MD, USA

Introduction: Congenital defects such as gastroschisis, omphalocele, congenital diaphragmatic hernia, or exstrophy involve a full thickness defect in the abdominal wall that requires a complex surgical approach, and often involve the use of permanent synthetic meshes which cause problems in the long term. An ideal approach to treating these conditions, as well as many acquired hernias or muscular defects, would use a synthetic mesh that serves as a scaffold for the creation of new tissue to fill the gap before the mesh dissolves. Our work with the PLLA nanofiber mesh suggests that it may serve that purpose and lead to a much more elegant method of treating these muscular defects.

Methods:   An abdominal wall defect was created in 54 rats (Rattus Norvegicus) followed by implantation of Vicryl (Polygalactin 910), Gortex (Preclude Vessel Guard), or Poly L-lactide (PLLA) Nanofiber mesh in an underlay fashion.  The abdominal wall was then harvested at 2, 6, or 12 weeks at which time a portion of the specimen underwent tensile strength testing and histologic processing. 

Results:  From a strength standpoint, there was no statistical significance between the strength of the Vicryl and PLLA Nanofiber.   While there was statistical significance amongst the three meshes at the 6 week endpoint with Gortex being stronger than both the Vicryl (p=0.033) and PLLA Nanofiber (p=0.041), the PLLA actually was stronger at the 12 week point. 

Conclusions:  This study demonstrated, to our knowledge, the first in vivo comparison of PLLA nanofiber mesh compared to two control meshes currently used in medical practice for repair of hernias and soft tissue defects.  The nanofiber mesh compared well with both control meshes and warrants further live tissue testing to further demonstrate potential efficacy in human usage as well as optimize physical and chemical modification parameters of this novel mesh.

 

63.06 A Histomorphometric Analysis of an Isogenic Model of Irradiated Distraction Osteogenesis

Posted on December 22, 2014

Y. Polyatskaya1, S. Deshpande1, A. Donneys1, S. Kang1, N. Nelson1, S. Deshpande1, P. Felice1, S. Buchman1  1University Of Michigan,Plastic Surgery,Ann Arbor, MI, USA

Introduction:  

Radiation therapy is known to be detrimental to bone and soft tissue repair, resulting in an unacceptably high incidence of devastating wound healing complications as well as the associated morbidity of late pathologic fractures and non-unions. Our global hypothesis is that the pathologic effects of radiation on bone formation and healing are mediated through a mechanism of direct cellular depletion as well as diminished function of the cells responsible for the generation and maintenance of osteogenesis. We sought to quantify the extent of this depletion via histomorphometric evaluation in an isogenic model of mandibular distraction osteogenesis for future analysis of cell-based therapies.

Methods:
20 male Lewis rats were randomly split into two groups, DO (n=10), XRT/DO (n=10). XRT underwent 5 day fractionated radiation of the left mandible at 7 Gy per day and were allowed to recover for two weeks. All groups underwent mandibular distractor placement. Groups were distracted at 0.3mm every 12 hrs to a 5.1mm (a critical-sized defect), and sacrificed on post-operative day 40. Coronal sections were stained using Gomori Trichrome. Quantitative histomorphometry was performed utilizing Bioquant. Statistical analysis was performed using student's t-test.

Results:
Radiation significantly decreased the ratio of Osteoid Volume / Total Volume (DO: 0.6774 ± 0.1193, XRT/DO: 0.4505 ± 0.08487, p=0.000), increased Bone Volume / Total Volume (DO: 0.3226 ± 0.11930 XRT/DO: 0.5495± 0.08487, p=0.000), and decreased Osteoid Volume / Bone Volume (DO: 2.7762 ± 2.31595, XRT/DO: 0.8578 ± 0.27674, p=0.017) . 

Conclusion:
A robust endogenous substrate is essential to distraction osteogenesis. Osteoid formation was impaired by radiation therapy and irradiated bone demonstrated hypermineralization. Thus, we demonstrated quantitative metrics of radiation-induced diminution of the matrix of bone, establishing an isogenic model for future remediation with cell- and tissue-based reconstructive therapies.      
 

63.07 Targeting KRAS in Pancreatic Cancer Using a Novel Method of Gene Silencing: U1 Adaptors

Posted on December 22, 2014

A. T. Tsang1,2, X. Yu1, R. Goraczniak5, M. Brenneman1,5, S. Gunderson3,5, D. R. Carpizo1,2,4  4Rutgers University,Department Of Pharmacology,Piscataway, NJ, USA 5Silagene Inc.,Hillsborough, NJ, USA 1Cancer Institute Of New Jersey,Division Of Surgical Oncology,New Brunswick, NJ, USA 2Robert Wood Johnson – Rutgers,Department Of Surgery,New Brunswick, NJ, USA 3Rutgers University,Department Of Molecular Biology And Biochemistry,Piscataway, NJ, USA

Introduction:

Activating mutations of the KRAS gene are key drivers of pancreatic cancer. However, despite decades of effort, the KRAS protein has proved refractory to small-molecule targeting. Many RNA interference-based studies targeting RAS have demonstrated therapeutic effects, but technical difficulties of delivery in vivo have impeded translation of this approach to the clinic. U1 Adaptors are synthetic oligonucleotides that enable the U1 small nuclear ribonucleoprotein complex to stably bind within the terminal exon of any chosen pre-mRNA target. This interferes with the obligatory polyadenosine tail addition step in mRNA maturation, resulting in selective destruction of the mRNA in the nucleus. The U1 Adaptor gene silencing mechanism is thus distinct from those of siRNA or antisense oligonucleotides, and offers important advantages for their use as therapeutic agents. In a proof-of-concept study, U1 adaptors targeting BCL2 and GRM-1 oncogenes effectively suppressed growth of human melanoma xenograft tumors in mice.

Methods:
We sought to translate this technology to target human KRAS in pancreatic cancer. We first screened a set of U1 Adaptors targeting human KRAS pre-mRNA at eight different positions within the terminal exon coding sequence and untranslated region. Adaptors were evaluated in vitro using the human pancreatic cancer cell line MiaPaca-2 (KRASG12C). KRAS mRNA was measured by quantitative PCR, and KRAS protein expression was determined using standard Western blot techniques. Cell growth inhibition assays were performed and viable cells were counted using flow cytometry over 12 days. Adaptors that gave the best in vitro results were then evaluated in MiaPaca-2 xenografts. For in-vivo delivery, Adaptors were complexed with nanoparticles linked to a tumor-targeting peptide, cRGD, and administered by tail vein injection twice weekly. Tumors were then evaluated for KRAS gene and protein expression.

Results:
U1 Adaptor screening revealed a range of KRAS gene silencing as measured by quantitative PCR. Candidate Adaptors 1, 2 and 3 reduced KRAS mRNA expression by 65%, 73% and 76% respectively; as effectively as an siRNA control. We demonstrated potent inhibition of MiaPaca-2 cell growth and knockdown of KRAS protein expression, using Adaptors modified with locked nucleic acid (LNA). We then evaluated Adaptors 2 and 3 in mice bearing MiaPaca-2 xenografts. We observed significant tumor growth inhibition, by as much as 68% using Adaptor 3 (p=0.0002), compared to the vehicle control by day 34. Tumors were then analyzed and found to have KRAS protein reduction.

Conclusion:
We have demonstrated that the U1 Adaptor method of gene silencing can be successfully applied to target human KRAS both in vitro and in vivo.  These results support the continued investigation of U1 Adaptor technology as a strategy for therapeutic targeting of RAS oncogenes.

63.08 Examining the pathogenesis of pouchitis using a tissue-realistic computational model: SEGMEnT_HPC

Posted on December 22, 2014

C. Cockrell1, S. Christley1, E. Chang2, G. An1  1University Of Chicago,Surgery,Chicago, IL, USA 2University Of Chicago,Medicine/Gastroenterology,Chicago, IL, USA

Introduction:  Pouchitis following proctocolectomy for ulcerative colitis is a significant source of morbidity, with long-term incidence rates of up to 95%.  The pathogenesis of pouchitis is a multiscale process involving the dysregulated intestinal inflammation, abnormal mucosal tissue response, and alterations in gut microflora due to stasis resulting from the anatomic configuration of the illeal pouch. We have previously developed the Spatially Explicit General-purpose Model of Enteric Tissue (SEGMEnT) to dynamically represent existing knowledge of the behavior of enteric epithelial tissue. Given that the progression of pouchitis and the spatial distribution of inciting stimuli are not homogenous throughout the pouch, anatomic scale simulations are required in order to plausibly simulate the clinical manifestations of the interplay of ileal and residual rectal cuff tissue with a dynamic microbiome. To achieve clinically relevant simulations we have implemented a supercomputing version of SEGMEnT, SEGMEnT_HPC, with the ability to generate anatomic-scale simulations of intestinal mucosa in response to inflammation.

Methods:  SEGMEnT_HPC is an expansion of our prior SEGMEnT model to a High Performance Computing (HPC) platform, incorporating gut epithelial cells, inflammatory cells and their effects on the extracellular matrix with consequent changes in crypt-villus morphology.  Luminal inflammatory stimuli are represented by the fecal microbiome as a dynamically flowing liquid. Simulation experiments were performed to investigate the role of fecal flow dynamics and host inflammatory potential on the development of anatomically realistic pouchitis, including anatomic transition zones at the proximal end of the pouch and the residual rectal cuff.

Results: SEGMEnT_HPC simulations suggested a putative role for Phosphotase and tensin homolog/phosphoinositide 3-kinase (PTEN/PI3K) as a key point of crosstalk between inflammation and morphogenesis, and demonstrated that differential flow dynamics associated with the creation of the ileal pouch promoted chronic inflammation due to fecal stasis in simulations in with impaired inflammatory suppression. We also noted the potential for the residual rectal cuff to “seed” the inflammatory propagation, seen in a heterogeneous continuum effect in terms of the anatomic patterning of metaplasia and inflammation in susceptible conditions. 

Conclusion: SEGMEnT_HPC can serve as an integrating platform for the study of inflammation in gastrointestinal disease by generating clinically relevant and anatomic scale phenotypes from cellular and molecular mechanisms. Simulations of pouchitis demonstrated a complex interplay between the inflammatory potential of the host, the effect of fecal stasis and alterations in the resident microbiome affected by the flow dynamics of the pouch and rectal cuff. We suggest that SEGMEnT_HPC will be a vital adjunct in our future exploration of this and other complex, multi-factorial processes

 

63.09 Memory T cell infiltration in hepatic colorectal adenocarcinoma metastases

Posted on December 22, 2014

J. V. Meyers1, S. Sen1, A. J. Tatar1, A. Contreras1, P. Srinand1, C. S. Cho1  1University Of Wisconsin,Section Of Surgical Oncology,Madison, WI, USA

Introduction:   Previous studies in primary colorectal adenocarcinoma have suggested that intratumoral infiltration by memory T cells may be strongly protective against the likelihood of metastatic disease dissemination.  The prevalence and prognostic significance of memory T cell infiltration within metastatic foci are unknown.  We hypothesized that memory T cell infiltration within resected hepatic colorectal metastases would be associated with favorable oncological characteristics.

Methods:   Frozen samples from resected hepatic colorectal adenocarcinoma metastases were obtained from a prospectively maintained and institutionally-approved tumor bank.  Thawed samples were fixed and analyzed by hematoxylin/eosin staining and by immunochemical analysis for expression of CD3 and CD45RO.  CD3 (T cell) and CD45RO (memory T cell) expression was quantified under low power magnification by two blinded observers using a two-tiered low versus medium/high grading system.  Clinicopathological and survival data were obtained from a prospectively-maintained institutional database.  The approximate risk of disease recurrence was stratified using the Memorial Sloan-Kettering Cancer Center Clinical Risk Score (CRS).  Comparison of categorical variables was performed using chi-square analysis, and differences in Kaplan-Meier estimates of overall and recurrence-free survival were compared using the log-rank test.

Results:  Samples from ten resected tumors were obtained for this preliminary analysis.  Eight tumors exhibited medium/high CD3 expression and 6 tumors exhibited medium/high CD45RO expression.  The prevalence of T cell infiltration was somewhat higher among patients with a CRS ≥ 2 (100%) than patients with a CRS < 2 (33%) (p=0.07).  The prevalence of memory T cell infiltration was higher among patients with a CRS ≥ 2 (86%) than patients with a CRS < 2 (0%) (p=0.03).  T cell and memory T cell infiltration were not associated with differences in overall or recurrence-free survival.

Conclusion:  There is a high prevalence of memory T cell infiltration within resected hepatic colorectal adenocarcinoma metastases.  Contrary to our hypothesis, intratumoral memory T cell infiltration alone is not obviously associated with differences in survival outcome, and its prevalence may be paradoxically higher in conditions of adverse tumor characteristics.  Ongoing studies will explore the hypothesis that intrametastatic memory T cell infiltration may exert a protective influence that mitigates the prognostic impact of adverse tumor characteristics.

 

63.10 Progenitor Rescue Model for the Investigation of Vascular Restenosis

Posted on December 22, 2014

B. W. Tillman1, T. D. Richards1, V. S. Donnenberg2  1University Of Pittsburgh Medical Center,Division Of Vascular/Department Of Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh Medical Center,Department Of Cardiothoracic Surgery,Pittsburgh, PA, USA

Introduction: Restenosis threatens the longevity of over half of all vascular interventions and circulating progenitor cells have been implicated for their role in this pathology.  This contrasts to the conventional paradigm of smooth muscle proliferation in restenosis.   Our laboratory has previously demonstrated significant reductions in restenosis after depletion of circulating progenitor cells.  Vascular injury signals are also believed to represent an important initial step in restenosis.  The exact role of vascular injury signals in restenosis, however, has been difficult to examine given overlapping roles in both progenitor mobilization and smooth muscle proliferation.  Previous reports have suggested reduced restenosis among mice with knockout of the vascular injury signal, matrix metalloproteinase 9, (MMP9KO).   We hypothesized that this finding may be related to impaired progenitor surge.  We further postulated that an approach of administering stem cell factor (SCF) may rescue progenitor surge even in the setting of impaired vascular injury signaling.

Methods: Transgenic MMPKO mice underwent wire injury of the femoral artery as part of an IACUC approved study. Blood was examined on post-operative day 1 using flow cytometry for CD34+/c-kit+ progenitor cells and results were compared to non-operative controls.  In a separate experiment, MMP9KO mice were treated with SCF for 4 days prior to flow cytometric analysis of blood specimens.   Significance of the results was determined using the Student’s t-test.  

Results: In contrast to our findings of progenitor surge after arterial wire injury in C57BL6 animals, our results in MMP9KO mice reveal no significant change among progenitors after wire injury compared to non-operative controls (P=0.74).   Administration of SCF demonstrated an average 2.3 fold increase among progenitors compared to saline treated controls (P=0.007)

Conclusion: In summary, our findings suggest that the vascular injury signal MMP9 is an essential factor in progenitor surge after vascular injury.  Increased progenitors after administration of SCF suggest this cytokine rescues progenitor surge even amidst deficiency of upstream MMP9.   This model presents a unique opportunity to examine the effects of progenitor cells on restenosis independent of the impact of vascular injury signals on vascular smooth muscle proliferation.  These results further support a model of vascular injury induced progenitor mobilization that ultimately contributes to restenotic pathology.

 

61.12 Surface Marker-Based Method for Isolation of Intestinal Subepithelial Myofibroblasts

Posted on December 22, 2014

H. A. Khalil1, N. Y. Lei1, P. Rana1, W. Nie1, J. Wang6, J. Yoo3, F. Wang2, L. Li2, M. G. Stelzner1,4, M. G. Martín6, J. C. Dunn1, M. Lewis5  1University Of California – Los Angeles,Surgery,Los Angeles, CA, USA 2Stowers Institute For Medical Research,Kansas City, MO, USA 3Tufts Medical Center,Surgery,Boston, MA, USA 4VA Greater Los Angeles Medical Center,Surgery,Los Angeles, CA, USA 5VA Greater Los Angeles Medical Center,Pathology,Los Angeles, CA, USA 6University Of California – Los Angeles,Pediatrics,Los Angeles, CA, USA

Introduction: The intestinal subepithelial myofibroblast (ISEMF) is an important component of the intestinal stem cell niche and a potential source of the Wnt-agonistic R-spondins. ISEMFs are traditionally identified by positive staining for α-smooth muscle actin (α-SMA) and vimentin and negative staining for desmin, but isolation is limited by lack of unique surface markers. We developed a surface marker-based approach for isolating ISEMFs.

 

Methods: ISEMFs were isolated from adult C57BL/6J mouse small intestine (n=4) and human duodenum (n=4) through digestion with collagenase D and dispase. Isolates were cultured in DMEM with 10% FBS, penicillin/streptomycin, epidermal growth factor, insulin, and transferrin, passaged weekly, and lysed for RNA. Deep sequencing was performed to assess the RNA expression of cell surface markers. Top surface markers were identified and paraffin-embedded mouse and human intestines were co-stained with antibodies against these surface markers as well as α-SMA. CD90 colocalized with α-SMA in the pericryptal region in mouse and human intestine and was used in the ensuing isolation algorithm. ISEMFs were isolated from digested whole mouse intestine (n=3) by fluorescence activated cell sorting using negative selection for annexin V and 7-AAD (apoptotic and dead cells), EpCAM (epithelial cells), CD31 (endothelial cells), and CD45 (hematopoietic cells). The resulting cells were sorted into CD90+ and CD90− groups. We analyzed unsorted and sorted cells for expression of α-SMA, vimentin, desmin, and R-spondin2 by qPCR.

Results: In culture, murine and human ISEMFs retained the characteristic positive staining for α-SMA and vimentin and negative staining for desmin. Deep sequencing of cultured mouse and human ISEMFs showed elevated expression of CD63, CD90, CD44, and CD248. This was confirmed by immunofluorescent staining of cultured ISEMFs, but staining mouse and human intestine for these markers showed α-SMA colocalization only with CD90. Isolation and sorting of digested mouse intestine by the negative and positive selection scheme described above resulted in α-SMA and vimentin enrichment in the CD90− group by 39.4±9.7 and 12.8±5.6 fold, respectively, relative to unsorted cells; the CD90+ group was enriched by 6.5±2.7 and 9.5±6.2 fold, respectively. Both CD90− and CD90+ populations were enriched with desmin (11.9±2.8 and 6.3±2.2, respectively, relative to unsorted cells). Expression of R-spondin2 was high in the CD90− and CD90+ populations (42.9±27.6 and 10.0±7.3, respectively, relative to unsorted cells).

Conclusion: Negative selection for epithelial, endothelial, and hematopoietic cells, with or without positive selection for CD90, yields cells that are enriched in the classical markers of ISEMFs and that highly express R-spondin2. Further work is needed to exclude desmin-positive smooth muscle cells.

61.13 Differential Mechanisms for Insulin-Positive Gene Expression in an Alpha Cell Line

Posted on December 22, 2014

I. Gaffar1,2, K. Prasadan2, P. Guo2, X. Xiao2, J. Wiersch2, C. Shiota2, G. Gittes2  1University Of Pittsburgh Medical Center,General Surgery,Pittsburgh, PA, USA 2Children’s Hospital Of Pittsburgh Of UPMC,Pediatric General And Thoracic Surgery,Pittsburgh, PA, USA

Introduction: Diabetes develops as a result of insufficient insulin production. One method to replace the beta cell mass would include the transdifferentiation of other endocrine cells into beta cells. Given that endocrine cells already have the machinery to produce and release hormones, it is plausible that a cell could be forced to express another hormone. The long-acting GLP-1 agonist, Exendin-4, has insulinotrophic properties in vitro. Exendin-4 has several important benefits with reduced risk for hypoglycemia and is an approved drug for the treatment for Type 2 Diabetes. Other studies have used Exendin-4 to treat neonatal mice with intrauterine growth retardation and prevented development of adult hepatic insulin resistance.  Additionally, the transcription factors Pdx1 and MafA have been demonstrated to play a key role in beta cell function and development. Our lab has previously used viral vectors to induce ectopic expression of transcription factors. In this study, we aim to show that alpha cells could become insulin producing cells.

Methods: Alpha TC cells (a transformed alpha cell line) were cultured with either Exendin-4 or an adeno-associated virus carrying Pdx1 and/or MafA. The viral vectors were constructed from recombinant rAAV plasmid vectors, pAAV-CMV-Pdx1, pAAV-CMV-MafA and pAAV-CMV-Pdx1-2A-MafA were constructed from pAAV-CMV-ZsGreen. AAV serotype 8 has been used in pancreatic cell transduction. AAV purification was performed with the rapid and simplified method.

Results: Our data show that alpha TC cells became insulin expressing cells when they were treated with a GLP-1 agonist, Exendin-4, or if they are transfected with an adeno-associated virus leading to ectopic expression of Pdx1 and MafA. We found that Exendin-4 induced alpha cell proliferation and some insulin expression, but most of the key markers of mature beta cells were not expressed. Further analysis show an increased expression of proliferation markers, but none of the mature beta cell markers were present in the cells exposed to Exendin-4. However, for the cells with forced expression of the transcription factors Pdx1 and/or MafA we saw robust expression of insulin and markers of mature beta cells.

Conclusion: In summary, our data suggests that there are two different possible mechanisms of transdifferentiation of alpha TC cells into insulin producing cells utilizing Exendin-4 to induced cell proliferation and immature beta cells, while ectopic expression of Pdx1 and MafA resulted in a more mature beta cell phenotype.

 

61.14 Bile Acids Lead to Induction of Intestinal Cell Death via Multiple Distinct Pathways

Posted on December 22, 2014

A. Roberts1, S. Papillon1, A. Dossa1, M. Frey3,4,5, H. Ford1,3,5, C. Gayer1,3  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 3University Of Southern California,Keck School Of Medicine,Los Angeles, CA, USA 4University Of Southern California,Pediatrics,Los Angeles, CA, USA 5Children’s Hospital Los Angeles,Developmental Biology And Regenerative Medicine,Los Angeles, CA, USA

Introduction: Necrotizing enterocolitis (NEC) is a severe neonatal intestinal disorder of premature infants. Animal models have shown how gavage of secondary bile acids can induce histologic changes in the intestinal epithelium that are identical to the damage seen in NEC. Intestinal bacteria convert primary bile acids (CA, CDCA) into more toxic secondary forms (DCA, LCA). Additional metabolism yields ursodeoxycholic acid (UDCA). The exact mechanism of cellular injury among different bile acids has not been elucidated. Apoptosis, necrosis, and necroptosis, a form of organized cell necrosis, are all potential mechanisms of cell death. We hypothesized that different bile acid metabolites will exert toxicity by separate mechanisms. 

Methods: IEC-6 and YAMC cell survival were assessed using an MTS-based assay. Z-VAD-FMK, a pan-caspase inhibitor, was used to measure apoptosis, along with measuring caspase-3 cleavage by western blot. Cell necrosis was quantified with an LDH-release assay. The role of necroptosis was assessed using necrostatin-1, a RIP1-kinase inhibitor, and confirmed with western blot of non-cleaved RIP3 protein. 

Results: Preliminary data in our lab shows that DCA and LCA are toxic to IEC-6 intestinal epithelial cells at lower doses than UDCA or primary bile acids. These results were also seen in a colon cell line (YAMC). Pre-treatment with Z-VAD increased survival in intestinal cells treated with DCA and LCA by 43% and 31% respectively, but did not change survival for primary bile acids at LD50 doses, indicating secondary bile acid-induced apoptosis. Western blot demonstrated increased caspase-3 cleavage with secondary bile acid treatment, confirming this mechanism. Bile acids conjugated with either glycine or taurine caused death by necrosis, with up to a 3-fold increase in LDH release. Unconjugated bile acids did not release a significant amount of LDH. Necrostatin-1 increased cell survival after treatment with UDCA, and western blot showed a 5-fold increase in RIP3 expression in these cells, both suggesting that UDCA toxicity occurs via necroptosis.

Conclusion: Our results show that bile acids lead to intestinal cell death via different mechanisms. Conjugated bile acids exert toxicity primarily by cell necrosis. Secondary bile acids are the most toxic, and induce apoptosis. UDCA in contrast, seems to cause necroptosis. The primary mechanism of toxicity for primary bile acids is not yet clear. Additionally, bile acids appear to have similar toxicity in the small intestine and colon. Interestingly, the colon normally has higher levels of secondary bile acids compared to the small intestine. This is the first report describing UDCA-associated necroptosis, and the first to compare intestinal epithelial cell toxicity from bile acids in different portions of bowel. Understanding the differential mechanisms of toxicity among specific bile acid metabolites may provide novel therapeutic targets in NEC, where secondary bile acids are more abundant.

61.16 Oncostatin M Receptor Deficiency Protects Against Sepsis In Older Mice

Posted on December 22, 2014

S. Y. Salim1, N. Al-Malki1, T. A. Churchill1, R. G. Khadaroo1  1University Of Alberta (CA),Division Of General Surgery, Department Of Surgery, Faculty Of Medicine & Dentistry,Edmonton, ALBERTA, Canada

Introduction:   Sepsis with concomitant multiple organ failure continues to be a clinical challenge, especially in the elderly who have increased morbidity and mortality. Oncostatin M (OSM) is part of the IL-6 cytokine family that has pleiotropic functions in hematopoiesis, immunologic and inflammatory networks. Levels of OSM have been previously shown to increase in patients undergoing septic shock, though the role and mechanism of OSM in sepsis remains elusive. Here we hypothesis that inhibition of OSM via OSM receptor (OSMR) deficiency, provides a survival benefit by modulating the inflammatory process in sepsis.

Methods:   Wild type (WT) litter-mates and OSMR knockouts (OSMR-/-), mice older than 50 weeks received intra-peritoneal injection of cecal slurry (CS). CS obtained from healthy WT C57BL/6 mice was prepared at LD30 of 1.3 grams of mice per mg of cecal contents. Mice were observed for 48 hours prior to peritoneal lavage and organ retrieval. Cytokine levels in tissue homogenates were measured using ELISA, while tissue were analyzed via myeloperoxidase (MPO) activity and histology.

Results:  Mortality rate was significantly higher in the WT mice (100%) compared to OSMR-/- mice (see figure). Clinical assessment showed differences between the strains occurring after 14 hours of CS injection. Cytokine analysis from tissue homogenates in both mice strains showed significant increase in IL-6, IL-10 and KC in lung and peritoneal lavage in CS compared to vehicle controls. Liver homogenates had significant increases in IL-6 and KC in both mice strains treated with CS compared to vehicle controls. Lung homogenates in CS treated OSMR-/- mice had significantly lower levels of IL-6 than WT. However, lung MPO activity was significantly higher in the OSMR-/- mice than WT mice treated with CS.

Conclusion:  This study shows that OSMR deficiency protects against septic shock in older mice. These mice had lower levels of lung IL-6 despite having greater neutrophil recruitment, thus indicating that OSMR deficiency results in decreased proinflammatory response. We speculate that OSM might play a deleterious role in distant organ failure in sepsis. Understanding the immunomodulatory activity of OSM in sepsis may provide novel therapeutic avenues in the future.

61.17 The Impact of Hemorrhagic Shock on Pituitary Function

Posted on December 22, 2014

A. A. Haider1, P. Rhee1, V. Pandit1, N. Kulvatunyou1, B. Zangbar1, M. Mino1, A. Tang1, T. O’Keeffe1, R. Latifi1, R. S. Friese1, B. Joseph1  1University Of Arizona,Trauma/Surgery/Medicine,Tucson, AZ, USA

Introduction:
Hypopituitarism after hypovolemic shock is well established in certain patient cohorts. Understanding the hormonal variations in trauma patients with hemorrhagic shock (HS) has been gaining focus. However; the effects of HS on pituitary function in trauma patients remains unknown. The aim of this study was to assess pituitary hormonal variations in trauma patients with hemorrhagic shock (HS). 

Methods:
Patients with acute traumatic HS presenting at our level 1 trauma center were prospectively enrolled. HS was defined as systolic blood pressure (SBP) ≤ 90 mm Hg and requirement of ≥ 2 units of packed red blood cell transfusion. Serum cortisol and serum pituitary hormones [vasopressin (ADH), adrenocorticotrophic hormone (ACTH), thyroid stimulating hormone (TSH), follicular stimulating hormone (FSH), and luteinizing hormone (LH)] were measured in each patient on admission and at 24, 48, 72,and 96 hours after admission. Outcome measure was: variation in pituitary hormones.

Results:

A total of 42 patients were prospectively enrolled with mean age 36.7±12.4 years , mean SBP 88±64.5 mm of Hg, and median injury severity score 26 [18-38] . The mean admission ADH level was 20.4±9.6 pg/ml, mean admission ACTH level was 42±38.4pg/ml, and mean admission cortisol level was 19.4±7.4 µg/dl. The pattern of pituitary hormones was variable over the 5 days (Figure 1). Four patients died within 24 hrs. Patients who died had higher mean admission ADH levels (p=0.01), higher mean admission ACTH levels (p=0.02), and lower mean admission cortisol levels (p=0.01) compared to patients who survived. On sub-analysis of patients who died after 24hrs (n=6), there was a significant decrease in the level of ADH (p=0.041) and cortisol (p=0.036).

Conclusion:
Acute hypopituitarism does not occur in trauma patients with acute hemorrhagic shock. The physiological response of the pituitary adrenal axis in HS was appropriate.  The pituitary hormone levels were variable over the 5 days. In patients who died there was a decreasing trend in the ADH and cortisol level.

61.18 Inhibition of a Hyperactive Glucocorticoid Receptor Fragment hGR-S1(-349A) by RU486

Posted on December 22, 2014

D. G. Greenhalgh1,2, K. Cho1,2, T. Green1, S. Leventhal1, D. Lim1, D. Greenhalgh1,2  1Shriners Hospitals For Children Northern California,Burns,Sacramento, CA, USA 2University Of California – Davis,Burns,Sacramento, CA, USA

Introduction: We have previously reported that a 118 amino acid fragment of the N-terminus of GR [hGR-S1(-349A)] which lacks ligand and DNA binding domains has a markedly enhanced response to steroids compared to the full-length GR (hGRa) alone.  How hGR-S1(-349A) enhances glucocorticoid activity is not known.  We sought to determine whether blocking classical glucocorticoid receptor pathway with RU486 would alter the enhanced activity of the altered hGR-S1(-349A). Since the fragment lacked both DNA and ligand binding domains we hypothesized that RU486 would not alter its activity.

Methods: Both the hGR and hGR-S1(-349A) were cloned into tsA201 cells and tested for glucocorticoid activity at various doses of hydrocortisone using our standard luciferase assay.

Results: hGR-S1(-349A) had the typical augmentation in response to increasing doses of hydrocortisone.  To our surprise, RU486 inhibited the activity of hGR-S1(-349A) suggesting a role for the hGR in its activity (figure).

Conclusion: hGR-S1(-349A) hyperactivity is inhibited by RU486 despite lacking DNA and ligand binding demands.  These findings suggest that hGR-S1(-349A) acts as a cofactor in conjunction with hGR to augment the glucocorticoid stress response.

 

61.19 CD13-Positive Selection of Human Adipose-Derived Stromal Cells Can Enhance Bone Formation

Posted on December 22, 2014

A. Luan1, C. Duldulao1, A. McArdle1, K. J. Paik1, M. T. Chung1, D. A. Atashroo1, E. R. Zielins1, R. Tevlin1, K. Senarath-Yapa1, T. Wearda1, S. Menon1, S. Shailendra1, M. Lee2, G. C. Gurtner1, D. C. Wan1, M. T. Longaker1  1Stanford University,Hagey Laboratory For Pediatric Regenerative Medicine, Department Of Surgery, Plastic And Reconstructive Surgery Division,Palo Alto, CA, USA 2University Of California – Los Angeles,Laboratory Of Biomaterials And Bioengineering, Division Of Advanced Prosthodontics, Department Of Bioengineering, Weintraub Center For Reconstructive Biotechnology,Los Angeles, CA, USA

Introduction:  The reduced ability of bone to regenerate past the age of 2 years in humans poses significant challenges for reconstructive surgeons. An ability to drive differentiation of adult stem cells isolated from human fat towards an osteogenic lineage (together with the use of biomimetic scaffolds) has resulted in techniques that offer great promise for treating bony defects, which present a significant biomedical burden. Human adipose-derived stromal cells (hASCs), however, are a heterogeneous population of multipotent cells. The aim of this study was to enrich for an osteogenic subpopulation within the heterogeneous hASC pool based on cell surface marker expression. This will ultimately offer a novel cell-based strategy for treating bone loss.

Methods:  We used a combination of single cell transcriptional analysis and Boolean implication networks to identify cell surface markers expressed on hASCs that are associated with upregulation of osteogenic gene expression. Using a bioinformatics analysis of our data, we identified CD13 as a potential target to enrich for osteogenic subpopulations of hASCs. To assess the osteogenic potential of CD13+ hASCs, we compared them in vitro to CD13– and unsorted hASCs. To evaluate osteogenesis in vivo, unsorted, CD13+, and CD13– hASCs were seeded onto a hydroxyapatite-poly(lactic-co-glycolic acid) scaffold and placed into critical sized calvarial defects of immunocompromised mice. Healing was measured over an eight week period using micro-computed tomography analysis. After 8 weeks, calvaria were harvested for histological assessment by pentachrome staining.

Results: Our results show that CD13+ hASCs demonstrate increased osteogenic differentiation in vitro as demonstrated by alkaline phosphatase staining at day 7 (**p<0.01) and extracellular matrix mineralization with Alizarin Red staining at day 14 (*p<0.05). In addition, gene expression analysis using qRT-PCR revealed an upregulation of RUNX2 and osteocalcin, early and late markers of osteogenesis, respectively. In vivo bone quantification showed increased bone formation in all three groups, with CD13+ hASCs demonstrating the greatest amount of healing. Bone formation was confirmed histologically.

Conclusion: The isolation of hASCs that express the CD13 surface marker enriches for a highly osteogenic subpopulation of hASCs. An ability to isolate an osteogenic subpopulation based on a single surface marker would allow for a clinically translatable method to isolate osteogenic populations for bone regeneration.

 

61.20 Fat Depot Biology in Microsurgical Autologous Breast Reconstruction Following Cancer

Posted on December 22, 2014

P. Singh1, P. Patel2, S. Fernandez2, P. Volden2, A. Spratt3, N. Jaskowiak1, M. Brady2, S. Conzen2, J. Park1  1University Of Chicago,Department Of Surgery,Chicago, IL, USA 2University Of Chicago,Department Of Medicine,Chicago, IL, USA 3University Of Chicago,The Division Of Biological Sciences,Chicago, IL, USA

Introduction:  The use of lower abdominal tissue for breast cancer reconstruction is a mainstay for reconstructive surgeons. Free flap (e.g. deep inferior epigastric perforator flap) reconstruction involves the microsurgical transfer of skin, subcutaneous fat and associated vasculature from the abdomen to the chest wall. As adipose tissue is now recognized as an endocrine organ that secretes a variety of fat-specific cytokines (“adipokines”) and lipid metabolites, the possible influence of transferred abdominal fat on breast cancer progression and recurrence is emerging as an important clinical question. While it is known that fat tissue displays differential adipokine secretion depending on anatomic location, little is known about subcutaneous abdominal versus breast fat characteristics. To begin to examine this, we hypothesized that breast fat from a cancer patient would exhibit increased metabolic activity and secrete more oncogenic adipokines when compared to subcutaneous abdominal fat. Our aims were to: 1) determine the metabolic and adipokine characteristics of abdominal versus endogenous breast fat depots in breast cancer patients undergoing reconstruction and 2) examine the impact of chest wall neovascularization on abdominal fat characteristics examined in Aim 1.

Methods:  Under an IRB-approved protocol, breast and subcutaneous abdominal fat were collected from 10 women undergoing mastectomy/reconstruction. Insulin signaling was measured in vitro using quantitative anti-phospho-Akt immunoblotting of adipose tissue protein lysates. In parallel, conditioned media was made by culturing minced fat from each depot in serum-free media for 8 hours, after which the media was applied to breast cancer cell lines and cell proliferation measured over time.

Results: Mean subject age was 60.7 (range 47–73) and mean BMI was 30.5 (range 25.9–36.7). Mean hemoglobin A1c was 5.6% (range 5.0–5.9%). Initial results suggest that breast fat demonstrated increased insulin sensitivity compared with abdominal fat. Conditioned media from both depots caused proliferation in MCF-10A (breast epithelial cell line) and MDA-MB-231 (triple-negative breast cancer cell line).

Conclusion: Initial data suggest that breast fat is more metabolically active than abdominal fat. Conditioned media studies indicate pro-proliferative factors are released from both depots. The conditioned media will also be analyzed for depot-specific adipokine signatures using antibody arrays. Three to six months following reconstruction, biopsies of the transferred subcutaneous abdominal fat will be done to determine if chest wall neovascularization alters insulin sensitivity and secretion of pro-proliferative factors compared to baseline abdominal tissue. Based on these studies, future studies comparing the vascularized transfer of alternative adipose depots (e.g. gluteal) could help identify the safest source of adipose tissue for post-mastectomy reconstruction.

 

62.01 Electrical Impedance as Non-invasive Metric for the Evaluation of Organ Quality in Ex-Vivo Lung Perfusion

Posted on December 22, 2014

D. M. Peterson1, S. M. Black3, S. Bennett3, C. Dumond3, D. Hayes2, R. S. Higgins3, B. A. Whitson3  1The Ohio State University,College Of Medicine,Columbus, OH, USA 2Nationwide Children’s Hospital (Columbus Children’s Hospital),Heart-Lung Transplant,Columbus, OH, USA 3The Ohio State University Wexner Medical Center,Surgery,Columbus, OH, USA

Introduction:

Ex-Vivo lung perfusion (EVLP) is a technique that enables the assessment of donor organs and the possibility of organ resuscitation potentially expand the donor pool. Currently, there is no defined method of assessing total organ edema, a condition that may contribute to primary graft dysfunction, poor gas exchange, and organ failure. We investigated the use of electrical impedance as a non-invasive measurement of extra-vascular water and organ edema in lungs undergoing EVLP. 

Methods:

Adult porcine lungs were procured and assessed in an IACUC approved protocol in a fashion similar to clinical practice. There were three perfusion groupings: Steen ®, saline and modified cell culture media (N=6). Standard procurement techniques were used with the pulmonary artery being antegrade flushed in situ.  At initiation of EVLP, the flow was increased to 40% of cardiac output with 1.5 Liters of perfusate slowly warmed to 37 ͦ C over 30 minutes. Lungs were ventilated at a tidal volume of 8 ml/kg, PEEP of 5 cmH2O, and respiratory rate of 8 bpm. Peak airway pressure, compliance, impedance, blood gas and extra vascular lung water (EVLW) were evaluated every 60 minutes. EVLW was evaluated using a PiCCO ® catheter in-line with the perfusion circuit. Impedance measurements were taken in the left lower lobe. 

Results:

The median pig weight was 40kg. Over the perfusion, lungs with the greatest injury had more significant changes in impedance values compared to those with less injury. Lungs perfused with Steen had less injury when compared to those with saline or modified cell culture media. As expected, impedance decreased in all lungs over time on the perfusion circuit. Decreases in impedance not only correlated with decreases in gas exchange (Delta PaO2/FiO2) but also preceded them alluding to a predictive component of impedance for changes in gas exchange. Decreases in impedance also correlated with decreases in lung compliance. Lungs perfused with Steen showed minimal overall change in impedance (38-134 Ω) compared to those perfused with modified cell culture (1425-1569 Ω) over a 4 hour perfusion period. 

Conclusion:

Electrical impedance offers a non-invasive method to measure lung tissue edema in a porcine EVLP model. Correlations between impedance, compliance and Delta PaO2/FiO2 values suggest impedance is a valid method for evaluating in lung edema over time during EVLP. Impedance offers increased sensitivity for lung water and appears to have predictive value for changes in gas exchange before the oxygenation changes. This technique could provide real time evaluation of donor lungs prior to transplantation. Further studies evaluating the use of impedance in other potential donor organs perfused ex vivo warrants investigation. 

62.02 A new method to measure intestinal secretion using FITC-Inulin in small bowel of rats

Posted on December 22, 2014

A. Munoz-Abraham1, G. Torres-Valencia1, T. Alfadda1, C. Jasinski1, R. Patron-Lozano1, M. I. Rodriguez-Davalos1, J. P. Geibel1  1Yale University School Of Medicine,Surgery – Transplant,New Haven, CT, USA

Introduction: Intestinal ischemia remains a major limitation in successful intestinal transplantation. Several animal intestinal ischemia models have been developed and used. However, rats remain as the most commonly used. The fragile balance of the intestinal mucosa relies on stable homeostatic mechanisms. To further understand the disruption of these mechanisms during ischemia/reperfusion injury, we have developed a new method to objectively measure osmotic changes in the intestinal lumen of rats that equate to injury.

Material and

Methods:  We used Sprague Dawley rats (401 to 442 grams).The rats were anesthetized and euthanized with isofluorane. 20 cm of distal ileum were taken and stripped from the mesentery. The intestinal lumen was flushed with regular HEPES solution (pH 7.408, mosm 297) to remove any remaining intestinal debris . The intestinal loops were then attached to two custom perfusion chambers that received a constant flow of regular HEPES solution. The chambers were submerged in a bath of deionized water at 37 C . At time 0, a known concentration of 3 ml of 0.001 mM FITC-Inulin was perfused into both lumens. Samples were collected and Relative Fluorescence Units (RFU) were measured using the NanoDrop 3300 Fluorospectrometer. The control intestine remained receiving a flow of regular HEPES, while the experimental intestine was bathed with a flowing solution of 10 µM Forskolin in a perfusion volume of 200 ml regular HEPES. Samples from the lumen were again collected from both intestines at times 25 and 35 minutes, and measured.

Results: A significant increase in luminal secretion was observed after the administration of the 10 µM Forskolin solution by observing the decrease in RFU units by almost half in the experimental intestine compared to the control.

Conclusion: It can be concluded that the use of FITC-Inulin can be an effective and objective method to measure fluid secretion or absorption in the small bowel, thus giving a more accurate estimate of the viability of the organ. By measuring the difference in RFU of a known solution after stop flow in the small bowel we demonstrated that absorption and secretion processes take place depending on the pathophysiological state of the organ. Absorption was observed when the intestine was maintained in physiological-like conditions, while secretion of large amounts of water into the lumen occurred when FSK was administered. In both cases the change in RFU using the method proposed gave accurate reproducible results with small sample variations  demonstrating the usesfulness of this model system for assessing intestinal viability.

 

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