5.05 Serum PROMININ-1: A Biomarker for Biliary Atresia–associated Fibrosis

Posted on December 22, 2014

M. Nguyen1,2, D. James1,2, N. Mavila1,2, S. Zhou1, L. Wang1, K. Wang1,2  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2Saban Research Institute,Developmental Biology And Regenerative Medicine,Los Angeles, CA, USA

Introduction:  

Biliary atresia (BA) is a severe neonatal liver disease caused by progressive fibro-obliteration of the biliary tree. BA is the leading indication for liver transplantation in children. Pathologically, BA is marked with intrahepatic ductular reactions and variable degrees of biliary fibrosis. We recently demonstrated the expansion of a population of myofibroblastic cells which co-express the stem cell marker PROMININ-1 (PROM1) and COLLAGEN-1α1 within regions of developing biliary fibrosis in experimental Rhesus rotavirus-induced BA (Mavila et al, Hepatology, 2014). In this study, we hypothesized that serum PROM1 levels in BA patients is a biomarker for severity of disease or liver fibrosis.

Methods:

Liver and blood specimens were obtained with parental informed consent from patients undergoing liver biopsy at CHLA under a study protocol approved by the Institutional Review Board. Ishak fibrosis scores were determined by two pathologists who were blinded to patient diagnosis and PROM1 expression levels. Serum levels were measured using a PROM1 ELISA assay kit (MyBioSource, San Diego). Statistical analyses of PROM1 serum levels were performed using Student's t-test. Quantitative Real-Time PCR (qPCR) was performed using cDNA with Light-Cycler Taqman Master (Roche) and probes from the Universal Probe Library (Roche Applied Science) using intron spanning and gene specific primers. Relative expression levels were calculated by Δ–Δ Ct method. 

Results:

Serum PROM1 levels correlated with liver tissue mRNA levels of PROM1 (R2 = 0.73). Serum PROM1 levels were significantly higher in patients with late-stage BA (n=4, p=0.02). BA patients who required liver transplantation expressed a greater than 2-fold increase in PROM1 serum levels compared to patients undergoing initial Kasai procedure (2.02±1.14 vs. 0.70±0.07 μg/ml, n=11, p=0.05). A similar trend in PROM1 levels was seen in liver samples, with a 14-fold increase in RNA expression in liver explants compared to native livers (n=3, p=0.05). Serum PROM1 levels also correlated with total bilirubin levels (linear regression analysis: R2 = 0.63). Ishak scores of ≥4 were associated with higher serum PROM1 levels than <4 (1.84 vs 0.69 μg/mL).

Conclusion:

Serum PROM1 levels correlate with the diagnosis of BA as well as the degree of biliary obstruction and fibrosis. Thus, serum PROM1 levels could be used potentially as a biomarker for BA-associated fibrosis.

 

5.06 Modifying the Embryonic Colonic Microenvironment Decreases Aganglionosis in Hirschsprung's Disease

Posted on December 22, 2014

A. J. Barlow-Anacker1, P. A. Trainor3, M. L. Epstein2, A. Gosain1,2  1University Of Wisconsin,Pediatric Surgery,Madison, WI, USA 2University Of Wisconsin,Neuroscience,Madison, WI, USA 3Stowers Institute For Medical Research,Kansas City, MO, USA

Introduction:
Hirschsprung’s disease (HSCR) is a congenital defect that results from the absence of ganglia along variable lengths of the bowel. These ganglia form from neural crest cells (NCC), a multipotent progenitor population that migrates over incredible distances to colonize the bowel. During migration NCC must survive, proliferate and differentiate to establish a functional enteric nervous system (ENS). Mutations in >10 different genes have been identified in HSCR patients; Endothelin receptor B (EdnrB) is one of the key players and NCC-specific deletion of EdnrB in mice (EdnrB-null) results in colonic aganglionosis, recapitulating the HSCR phenotype. The inability of NCC to colonize the entire colon in these mice has been demonstrated to result from temporal changes within the gut microenvironment. Identification of the molecular profile of the non-permissive colonic microenvironment in EdnrB-null animals may delineate how NCC interact with and subsequently colonize the gut microenvironment.

Methods:
RNA was isolated from the terminal colon of E14 EdnrB-null and wild type (WT) embryos and used to probe Affymetrix GeneChip mouse genome microarrays. A nine-way independent pair-wise comparison between EdnrB-null and WT tissue was performed and log2 mean values equivalent to a gross fold change of ± 1.5 used as a threshold to identify differentially expressed genes. Quantitative PCR was then performed to validate the differential gene expression. E13 colonic explants were cultured in the presence of a blocking peptide to Laminin and the effect on NCC advancement was determined. 

Results:
Microarray analysis of the molecular profile of WT and EdnrB-null terminal colonic tissue identified a total of 131 differentially expressed genes. 57 of these genes were up-regulated while 74 were down-regulated. Quantitative PCR confirmed the microarray results. Laminin, a component of the extracellular matrix, was markedly up-regulated in EdnrB-null colon. Ex vivo culture of E13 colon in the presence of a laminin blocking peptide resulted in almost complete NCC colonization of the colon.

Conclusion:
We have identified the molecular profile of EdnrB-null colonic tissue that is non-permissive to NCC advancement along the colon beyond E14. Competitive blockade of laminin, shown to be up-regulated in EdnrB-null vs WT colonic tissue, results in the increased advancement of NCC along the gut ex vivo. These results are the first demonstration of increased colonization by NCC of the non-permissive colon in a mouse model of HSCR. Future approaches directed at decreasing the length of aganglionosis may reduce long-term morbidity in HSCR patients. 
 

5.07 Growth and Biomechanics after Composite Biological Patch Repair in Rat Model of Diaphragmatic Hernia

Posted on December 22, 2014

G. P. Liao1, R. A. Hetz1, C. J. Corkins1, T. G. Hughes1, H. Xue1, Y. Li1, K. P. Lally1, C. S. Cox1  1University Of Texas Health Science Center At Houston,Pediatric Surgery,Houston, TX, USA

Introduction:
Tissue engineering for congenital diaphragmatic hernia (CDH) repair seeks to improve biomechanical compatibility while reducing device failure. Decellularized scaffolds have the potential of regenerating the structure and function of their native tissue over commercially available matrices from other tissues, but lack immediate strength. Our lab explored using patches constructed with decellularized rat diaphragm combined with porcine bladder matrix in a preclinical rodent model for potential translational repair of CDH diagnosed in utero.

Methods:
A composite patch made of decellularized rat diaphragm combined with porcine bladder matrix was implanted in rats with surgically created left sided diaphragmatic hernias (n=5). Control rats received either decellularized diaphragm patch alone (n=9) or porcine bladder matrix patch alone (n=5). At four months post implantation, the rats were weighed and their diaphragms explanted and subjected to physiologic testing of maximum force generation and modular tensile strength.

Results:
Rats repaired with the composite patch gained 124+/-6 grams, compared to 100+/-2 grams with the decellularized diaphragm alone (p=0.02) and 77 +/-4 grams with the porcine bladder matrix alone (p=0.003). Pulse stimulation of explanted hemidiaphragms repaired with the composite patch generated 71+/-18% of the contralateral hemidiaphragm force compared to 47+/-8% with the porcine bladder matrix patch alone (p=0.21) and 35+/-5% with the decellularized rat diaphragm patch alone (p=0.03). Composite patch hemidiaphragms had closer to normal (1 MPa) modular tensile strength, at 1.2+/-0.1 MPa than hemidiaphragms repaired with the decellularized rat diaphragm patch 1.8+/-0.2 MPa (p=0.01) and than porcine bladder matrix 1.8+/-0.3 (p=0.08).

Conclusion:
Diaphragmatic defects repaired with composite biological patches that combine decellularized native tissue and commercially available matrices improve diaphragmatic biomechanical function and modular tensile strength and are associated with improved weight gain.

5.08 A Novel Method of Human Adipose-derived Stem Cell Isolation With Resultant Increased Cell Yield

Posted on December 22, 2014

R. Tevlin1,2, A. Mc Ardle1,2, E. Seo2, M. T. Chung1, K. Paik1, E. R. Zielins1, D. Atashroo1, M. S. Hu1, G. G. Walmsley1,2, S. Li1, C. K. Chan2, D. C. Wan1, M. T. Longaker1,2  1Stanford University,Plastic Surgery,Palo Alto, CA, USA 2Stanford University,Institute for Stem Cell Biology and Regenerative Medicine,Palo Alto, CA, USA

Introduction:

Adipose tissue represents an abundant and easily accessible source of multipotent cells which may serve as excellent building blocks for tissue engineering. We have developed a new protocol for isolating adipose-derived stromal cells (ASC) from human lipoaspirate. The aim of this study was to compare our new method for ASC isolation to standard protocol (Zuk et al, 2002).

Methods:

Human ASC isolation was performed using two methods to compare cell yield, cell viability and cell proliferation. The osteogenic and adipogenic potential of ASCs isolated using both protocols was assessed in vitro and gene expression analysis was performed. To assess the ability of isolated ASCs to generate bone in vivo, we created critical sized calvarial defects in mice which we treated with ASCs loaded onto HA-PLGA scaffolds.

Results:
We have developed a novel protocol which results in a 10-fold increased yield of ASCs compared to conventional technique. Resulting ASCs harvested using our new protocol demonstrate significantly increased cell viability and proliferation in vitro (p<0.05). Additionally, ASCs isolated using our new protocol demonstrated significantly enhanced osteogenic and adipogenic differentiation capacity in vitro (p<0.05). ASCs harvested using our new protocol have an equal ability to heal critical sized calvarial defects in mice.

Conclusion:

We have developed a protocol that maximizes the yield of ASCs derived from lipoaspirate. We demonstrate that ASCs harvested using this method have an increased osteogenic and adipogenic potential in vitro and are equivalent to ASCs isolated using the conventional protocol in terms of their ability to generate bone in vivo.

 

Reference: 

Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H et al. (2002). Human adipose tissue is a source of multipotent stem cells. Molecular biology of the cell 13(12):4279-4295.

 

5.09 Reconstitution of Bronchoalveolar Progenitors in Tissue Engineered Murine Lung

Posted on December 22, 2014

A. Trecartin1, R. Spurrier1, D. Warburton1, B. Driscoll1, M. Hiatt1, T. Grikscheit1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:  Lung disease is the fourth leading cause of death in the US and remains a significant cause of morbidity and mortality in pediatric and adult surgical patients. Lung stem cells and critical signaling from supporting mesenchymal cells are implicated in the pathogenesis of and recovery from lung injury.  Bronchoalveolar stem cells (BASCs) appear to play a critical role in self-renewal and repair, differentiating into both club cells and type II alveolar epithelial cells in vitro. Definitive demonstration of BASC differentiation in-vivo has yet to be performed. A unique feature of BASCs is their expression of both surfactant protein C (SP-C) and the club cell marker CC10. To our knowledge there has been no previous successful development of tissue-engineered lung (TELu) containing these progenitors.

Methods:  Lung organoid units (LOU) were prepared from actin-GFP murine lung tissue in a variation of a previously published protocol to generate OU from small intestine and were loaded onto poly-glycolic acid/poly-L lactic acid scaffolds. These scaffolds were then implanted subcutaneously into C57BL/6 hosts. Four weeks later the implants were harvested, preserved in paraffin, sectioned and prepared for immunofluorescence staining.  A section of native actin-GFP mouse lung tissue was stained as a control. The slides were incubated with primary antibodies for T1α, SP-C, and CC10. After washing, the secondary antibodies labeled with Alexa Fluor® 488 for T1α’s primary antibody host, Alexa Fluor® 568 for SP-C, and Alexa Fluor® 680 for CC10 were placed. These were subsequently imaged in green, red, and far-red channels respectively.

Results: Both native lung and TELu show positive staining for T1α and SP-C indicating the presence of type I and II alveolar epithelial cells respectively. In some fields in TELu, clusters of multiple cells are identified with positive staining for both SP-C and CC10, indicating presence of bronchoalveolar stem/progenitor cells (BASCs).

Conclusion: TELu is a novel in-vivo model in which multiple lung epithelial subtypes are identified, including Type I and II alveolar epithelial cells and bronchoalveolar stem cells. BASCs are not identified in native lung in the larger clusters imaged in TELu, but usually are found at the bronchoalveolar junctions.  Which may make this in-vivo model useful for better understanding the stem/progenitor cell functions that underpin regeneration of lung injury.

 

5.10 Regulation of WNT signaling in craniofacial convergence and extension morphogenesis.

Posted on December 22, 2014

L. J. Rochard1, S. J. Monica1, Y. Kong1, M. J. Grimaldi1, E. C. Liao1  1Massachusetts General Hospital,Center For Regenerative Medicine,Boston, MA, USA

Introduction: Orofacial clefts, such as cleft lip and palate (CL/P), are the most common congenital anomalies with a frequency of 1 in 700 births. Several genetic causes have been described and one of the main signaling pathways involved is WNT. However the precise role of WNT pathway in morphogenesis of the palate remains poorly understood. WNT proteins are secreted factors where intracellular chaperon and export is regulated by WLS. The WLS gene is located on the short arm of the chromosome 1 and several deletions of this region are associated with craniofacial anomalies. 

Methods: We utilize a zebrafish wls mutant to genetically dissect the role of WNT in palate formation. Expression analysis of wls, neural crest and Wnt signaling pathway genes were detected by RNA in situ hybridization during embryogenesis. Craniofacial structures in wls mutants were delineated using Alcian blue staining. Cell lineage, apoptosis and proliferation assays were performed to determine effect of wls mutation on these cellular behaviors.

Results:
wls gene expression was detected in the oropharyngeal epithelium, juxtaposed to the palate chondrocytes, which express extracellular ligand frzb. The epithelium also expresses secreted ligand wnt9a, and receptor gpc4. In this mutant, the wls protein is truncated, abrogating the WNT secretion. Phenotype analysis demonstrated that the palate is malformed, shorter in the anteroposterior axis and wider in the transverse dimension. On the cellular level, the palate chondrocytes are rounded instead of disc-like, and the cells fail to intercalate and organize in a single layer as in wildtype. We also find that migration and proliferation of chondrocytes are normal. Molecular analysis revealed that expression of wnt9a is unaffected with loss of wls, but expression domain of frzb is expanded proximally, while it should be restricted to the proliferative front. Taken together, the palate malformation is due to failure in convergence and extension mechanisms of morphogenesis, where regulation of frzb expression is disrupted. 

Conclusion:Analysis of wls mutant demonstrates that WNT pathway components are coordinated to regulate convergence and extension mechanisms. Taking a genetic approach, wnt9a and frzb mutants have been generated via CRISPR to test genetic interaction between these WNT components. We also generated fluorescent epitope tagged proteins of wls, frzb and wnt9a to directly visualize intracellular and extracellular trafficking of these components during cellular morphogenesis. These studies will gain insight into the molecular basis of craniofacial development and highlight the utility of the zebrafish model in complementary genetic and developmental analyses.

 

5.11 Scarless Surgery: Are We There Yet?

Posted on December 22, 2014

C. M. Moles1, S. Balaji1, N. Han1, R. Ranjan1, A. Shaaban2, P. Bollyky3, T. M. Crombleholme4, S. G. Keswani1  1Cincinnati Children’s Hospital Medical Center,Laboratory for Regenerative Wound Healing,Cincinnati, OH, USA 2Cincinnati Children’s Hospital Medical Center,Center For Fetal Cellular And Molecular Therapy,Cincinnati, OH, USA 3Stanford University,Palo Alto, CA, USA 4Children’s Hospital Colorado,Aurora, CO, USA

Introduction:  More than 100 million patients acquire scars annually in the developed world. Currently, there is no available anti-scar therapy. The fetal response to injury is regenerative and can serve as a roadmap to achieve postnatal scarless wound healing. Fetal wounds have high levels of IL-10, and viral over-expression of IL-10 results in postnatal regenerative wound healing. We hypothesize that delivery of IL-10 in a more clinically translatable sustained release hydrogel (HH10) can result in regenerative healing in postnatal wounds. 

Methods:  4mm full thickness dorsal wounds in C57BL/6J mice were created (n=20). Wounds were harvested at 28 days post wounding and histological evaluation (H&E) and capillary density (CD 31+ caps/HPF) of uninjured skin and scars were performed. Observed differences were used to establish 5 quantifiable scar parameters. Following development of a novel scar scale, in vivo wound healing experiments testing the efficacy of HH10 were performed. Treatment groups include HH10, Gel Control, Lentiviral IL-10 (LV-IL-10) or PBS (n=4/group). HH10 is a scaffold made of 2:1:1 (hyaluronan conjugated with heparan sulphate, type-I collagen and polyethylene glycol diacrylate) and IL-10 (800ng/wound). Data presented as mean+/-SD, p-values by ANOVA or T-test.

Results: Histologic analysis demonstrates significant differences between uninjured skin and scar in epidermal height, nuclear orientation of the basal keratinocytes, scar area, dermal appendages and vascular density (column A vs. B; p<.01). HH10 treatment yields significantly improved scar parameters compared to characteristic scar in PBS wounds (column D vs. B; p<.01). Scar assessment reveals HH10 and viral over-expression of IL-10 are equally potent in achieving attenuation of scar (column D vs. C; p=ns). HH10 restores epidermal and dermal scar parameters to the levels observed in uninjured skin (column D vs. A; p=ns). Gel treatment without IL-10 improves wound healing compared to PBS (column E vs. B; p<.05), but not to levels seen with HH10 or LV-IL-10 (column E vs. D or C; p<.05).

Conclusion: 

Using a novel quantifiable method to assess scars histologically, HH10 results in restoration of epidermal and dermal parameters to the levels observed in unwounded skin, the benchmark of regenerative healing. HH10 obviates some of the translatable concerns with IL-10 gene therapy, and has broad potential applications. The therapeutic benefits extend beyond the cosmetic benefit, and may apply to any disease characterized by excessive fibroplasia.

 

5.12 Nanofiber Enhanced Endothelialization of Decellularized Cardiac Tissue

Posted on December 22, 2014

R. S. Hennessy1, S. Jana1, M. Helder1, A. Lerman1  1Mayo Clinic,Rochester, MN, USA

Introduction: Endothelization of decellularized heart valves is required to lower thrombogenicity risk and decrease inflammation of tissue-engineered valves.  However, conventional methods of endothelization do not show positive results and can take upwards of 2 weeks to result in a monolayer. These cells are often statically adaptive and do not adhere firmly to the 3-D structure of the heart valve cusp.  We hypothesized that the addition of a cell receptive biodegradable material in nanofiber form could increase endothelial cell viability, proliferation, and adherence. 

 

Methods: Porcine aortic valve cusps were decellularized with 1% sodium dodecyl sulfate.  After washing and sterilization, the cusps were covered with polycaprolactone (PCL, 9 wt% solution) nanofibers through electrospinning system at a voltage of 17.5 kV and with a distance between the spinneret and collector of 15 cm. The encased cusps and cusps without a nanofiber layer (bare cusp) were then seeded (106 cells/ml) with blood outgrowth endothelial cells (BOECs) and cultured statically with periodic observation. The morphology of the nanofibers and cells on cusps were analyzed with scanning electron microscopy (SEM).  The cells on cusps were also stained with Hoechst and imaged with a confocal microscope. Cell counting was performed on confocal images manually and areas covered by cells were calculated with ImageJ software.

 

Results:The diameter of the electrospun nanofibers was ~200 nm (Fig. a). The bare cusps were covered with an endothelial cell-layer at 14 days of culture (Fig. b) whereas nanofiber-layered cusps formed a monolayer at 4 days (Fig. c).  Endothelial cells on bare cusps under SEM had a globular profile and cell-cell junctions were sparse.  On nanofiber cusps, SEM showed elongated endothelial cells and the presence of cell-cell junctions.  The densities of nuclei shown in Figures d and e obtained through confocal imaging confirmed higher cell proliferation and growth on nanofibrous covered cusps compared to naked cusps. These results are further confirmed by cell number and cell-covered area on these cusps (Fig. f).
 

Conclusion:A nanofibrous layer made of PCL showed efficacy in improving endothelial cell propagation and growth. The nanofibrous layer also influenced endothelial cells to develop an almost continuous monolayer in 4 days as compared to 14 days in the absence of the nanofibrous layer.  Thus, it can be concluded that a biodegradable PCL polymeric nanofibrous layer is helpful to achieving a more robust endothelial layer.

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.  

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