43.07 DPR Decreases TLR4 and MYD88 Expression in the Lung after Resuscitated Hemorrhagic Shock

Posted on December 22, 2014

M. A. Eid2, P. J. Matheson1,2,3, C. D. Downard1,2, R. N. Garrison1,2,3, J. W. Smith2,3  1Robley Rex Veterans Affairs Medical Center,Louisville, KY, USA 2University Of Louisville,Surgery,Louisville, KY, USA 3University Of Louisville,Physiology & Biophysics,Louisville, KY, USA

Introduction: Acute Lung Injury (ALI) leading to Adult Respiratory Distress Syndrome (ARDS) is an early sign of multiple organ dysfunction (MOD) after hemorrhagic shock (HS).  Gut-derived pro-inflammatory factors via mesenteric lymph initiate systemic inflammatory response (SIRS), possibly via lung toll-like receptor 4 (TLR4). Adjunct Direct Peritoneal Resuscitation (DPR) following HS mitigates the inflammatory response.  Deficiencies in downstream pathways from TLR4 (i.e., MYD88 and TRIF) attenuate SIRS by an unknown mechanism.  We hypothesized that DPR would improve lung function in resuscitated HS (HS/CR) by altering levels of TLR4 and 2nd messengers MYD88/TRIF.

 

Methods:   Anesthetized Sprague-Dawley rats were randomly assigned to groups (n=8/group):  1) HS/CR (HS=40% MAP for 60min, CR=shed blood + volumes NS); 2) HS/CR+DPR at time of CR;  3) HS/CR+DPR(120) at 120min post-resuscitation (postRES); 4) Sham (no HS, no CR, no DPR); 5) Sham + DPR at CR; and 6) Sham+DPR(120).  All groups were followed for 4hr postRES.  ELISA was used to measure lung TLR4, MYD88, and TRIF as well as mesenteric lymph and serum LPS.

Results:  HS/CR increased LPS and TLR4 and MYD88 expression in the lung compared to Shams but did not alter TRIF (see Figure).  HS/CR+DPR decreased TLR4 and MYD88 levels but did not alter TRIF levels.  Delayed addition of DPR (HS/CR +DPR(120)) had a similar effect.

Conclusions:  Gut-derived mediators of systemic inflammation can be modulated by peritoneal resuscitation with hypertonic peritoneal dialysis solution to prevent activation of lung inflammatory processes.  These benefits occur when DPR is given immediately at the time of conventional resuscitation or when DPR is delayed by 2 hours after the start of conventional resuscitation.

43.08 MyD88 Inhibition in Limb Ischemia Increases Myofiber Size and Perfusion in TRIFKO Mice

Posted on December 22, 2014

J. Xu1, X. Cui1, E. Tzeng1, U. Sachdev1  1University Of Pittsburgh,Vascular Surgery,Pittsburgh, PA, USA

Introduction: We have shown that mice with genetically absent MyD88, a critical mediator of TLR4 signaling, showed less ischemic damage and inflammation than controls after femoral artery ligation.  Surprisingly, mice lacking TRIF, another important mediator of TLR4 signaling demonstrated necrosis rather than protection at the same time point. These earlier findings suggested that MyD88 and TRIF played complimentary roles in the recovery from limb ischemia. In order to further test this theory, we treated control C57B6 mice as well as TRIFKO with a peptide inhibitor of MyD88 to test the hypothesis that unopposed MyD88 inflammation in TRIFKO mice hindered muscle recovery after femoral ligation.

Method: C57B6 mice and TRIFKO mice were injected with 25 micrograms of peptide inhibitors to MyD88 or a nonspecific target one hour before the initiation of femoral artery ligation on the right hindlimb. On the left, the femoral vessels were exposed but not ligated. Laser Doppler Perfusion Imaging (LDPI) was performed after one week to assess perfusion recovery, expressed as a ratio of right to left flow. H and E was performed on ischemic muscle samples and the average area of each regenerating myocyte, identified by centrally located nuclei, was compared among groups.

Results:  TRIFKO mice treated with a MyD88 inhibitor demonstrated an improvement in perfusion by LDPI 7 days after ligation (Figure 1). Neither control C57B6 mice nor mice treated with control peptide inhibitor demonstrated similar improvements in perfusion to the ischemic limb at the same time point.  Similarly, the mean size of regenerating myocytes was significantly higher in TRIFKO mice treated with MyD88 inhibitor but not control inhibitor (295.12 ± 14.98 vs. 235.00 ± 14.72 micrometers; MyD88 inhibitor vs. control inhibitor, p=0.038, N=3-4/group). Similar improvements were not demonstrated in C57B6 mice treated with the MyD88 inhibitor.

Conclusion: Our data suggest that in the absence of TRIF, MyD88 activity may compromise muscle regeneration after ischemia.  Blocking its activity with a peptide inhibitor improves perfusion recovery and increases regenerating myofiber size after one week.  These results further support the hypothesis that TRIF attenuates MyD88 activity, acting to balance the deleterious inflammatory effects of intact MyD88 signaling in limb ischemia.  

43.10 Adeno-Thioredoxin- 1 Delivery Improves Neovascularization in a Murine Model of Hind Limb Ischemia.

Posted on December 22, 2014

M. T. Rishi1,2, I. A. Shaikh1,2, V. Selvaraju1, M. Thirunavukkarasu1, J. Palesty2, N. Maulik1  1University Of Connecticut Health Center,Molecular Cardiology And Angiogenesis Laboratory, Department Of Surgery,Farmington, CT, USA 2Saint Mary’s Hospital,Stanley J. Dudrick Department Of Surgery,Waterbury, CT, USA

Introduction: There is a growing interest in developing novel therapeutic modalities at a molecular level for the treatment of peripheral arterial disease (PAD). We have previously shown that Thioredoxin -1 (Trx-1), which is a cytosolic 12-kDa redox protein, enhances neovascularization and reduces ventricular remodeling during chronic myocardial infarction. Here we aim to investigate the role of this protein in a murine hind limb ischemia (HLI) model.

Methods: Adult 8-12 week old C57Bl/6 mice were divided into two groups: (1) adeno-thiroedoxin-1 gene treatment group (Ad-Trx-1) and (2) control group (Ad-LacZ). The mice in Ad-Trx-1 group underwent right femoral artery ligation to create a murine model of HLI. Immediately after surgery, adeno-thioredoxin -1 in a concentration of 1×10⁹ PFU was injected in both semimembranosus and gastrocnemius muscles of the right leg whereas the left leg was used as an internal control. The mice in Ad-LacZ group also underwent right femoral artery ligation but instead received similar concentration of Ad-LacZ at the same muscle sites. The two groups underwent serial laser doppler imaging (LDI) pre-operatively and post-operatively for 28 days to assess hind limb perfusion. Immunohistochemistry and ELISA were performed on post-operative day 4 to determine the expression for various key angiogenic proteins. 

Results: Mice in the Ad-Trx-1 group showed a significantly increased perfusion ratio on postoperative day 21  [0.893±0.067 (n=10) vs. 0.593±0.065 (n=10); p<0.05] and day 28 [0.908±0.081 (n=08) vs. 0.660±0.057 (n=10); p<0.05] [Figure 1] and a higher motor function score on post-operative day 7 [3.1±0.233 (n=10) vs. 2.1±0.276 (n=10); p<0.05], day 14 [3.9±0.233 (n=10) vs. 2.8±0.249 (n=10); p<0.05], day 21 [4.9±0.1 (n=10) vs. 3.7±0.213 (n=10); p<0.05] and day 28 [4.875±0.125 (n=8) vs. 3.8±0.249 (n=10); p<0.05] as compared to mice in Ad-LacZ group. Four days after femoral artery ligation, Ad-Trx-1 group showed increased Vascular Endothelial Growth Factor (VEGF) expression by immunohistochemical analysis and ELISA [38.48±7.165 (n=4) vs. 21.70±2.560 (n=5); p<0.05] as compared to Ad-LacZ group. Ad-Trx-1 group also showed increase expression of Flt-1 and Angiopoietin -1 proteins as compared to Ad-LacZ group by immunohistochemical analysis. 

Conclusion: Taken together, our study demonstrates that the adeno-thioredoxin-1 gene delivery enhances blood perfusion and increases angiogenic protein expression in a murine hind limb ischemia model. We hope that this molecule can be a future potential target for clinical trials and subsequently drug therapy in peripheral vascular disease management.

 

 

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.

 

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

Posted on December 22, 2014

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

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

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

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

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

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

Posted on December 22, 2014

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

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

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

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

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

4.18 THROMBELASTOGRAPHY PERFOMRED WITHOUT AN ACTIVATOR ENHANCES DETECTION OF FIBRINOLYSIS

Posted on December 22, 2014

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

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

 

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




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

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

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

Posted on December 22, 2014

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

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

Methods:

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

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

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

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

4.16 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.

4.15 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.

4.13 The Anti-Cholinergic Pathway Protects Against Intestinal Barrier Dysfunction and DAMPs Release

Posted on December 22, 2014

M. E. Diebel1, D. M. Liberati1, L. N. Diebel1  1Wayne State University,Michael And Marian Ilitch Department Of Surgery,Detroit, MI, USA

Introduction: Intestinal barrier injury occurs following major trauma and leads to an  intestinal inflammatory response and subsequent remote organ dysfunction. This response may be modulated by either vagal nerve stimulation or pharmacologic stimulation of the alpha7-cholinergic receptor (nAChR) anti-inflammatory pathway. The downstream mediators in this pathway are relatively unknown. Gut injury is also associated with the release of endogenous damage associated molecular patterns (DAMPs) which may modulate shock induced organ dysfunction. The impact of stimulation of the intestinal cholinergic anti-inflammatory pathway on DAMPs release and resultant tissue injury was studied in vitro.

Methods: Intestinal epithelial cell (IEC-6) monolayers were subjected to hypoxia-reoxygenation (H/R) challenge. Cell subsets were treated after hypoxic challenge with nicotine or AR-R17779, a specific nAChR agonist. Nuclear factor kappa light-chain-enhancer of activated B cell (NFkB) activation was determined by ELISA and IEC monolayer integrity was indexed by permeability to an FITC-dextran probe (4,000 mw; FD-4). DAMPs production was indexed by high mobility group box 1 (HMGB-1) (western blot) and mitochondrial DNA (coxIII using RT-PCR) release. Human pulmonary microvascular endothelial cells (HMVEC) were then co cultured with IEC culture supernatants and monolayer permeability and ICAM-1 expression determined.

Results: mean ± S.D., N = 4 for each group

Conclusion: Pharmacologic stimulation of the nAChR pathway protected against H/R induced intestinal barrier derangement, NFkB activation and DAMPs release. Decreased IEC mediated DAMPs release was associated with protection against lung microvascular injury and ICAM-1 expression in this in vitro study. Modulation of this pathway may be helpful in the clinical setting.

 

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

Posted on December 22, 2014

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

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

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

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

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

 

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

Posted on December 22, 2014

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

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

 

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

 

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

 

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

 

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

Posted on December 22, 2014

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

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

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

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

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

 

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

Posted on December 22, 2014

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

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

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

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

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

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

4.06 An Accurate Method For Predicting Death From Sepsis

Posted on December 22, 2014

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

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

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

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

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

 

4.07 The Effects of Exercise on Soleus Function in Severe Burn with Muscle Disuse Atrophy

Posted on December 22, 2014

M. R. Saeman1, K. DeSpain1, M. Liu1, B. Carlson1, L. A. Baer2, J. Song1, C. E. Wade2, S. E. Wolf1  1University Of Texas Southwestern Medical Center,Surgery,Dallas, TEXAS, USA 2University Of Texas Health Science Center At Houston,Surgery,Houston, TEXAS, USA

Introduction:
Muscle loss is a known sequela of severe burn and critical illness that increases the risk of complications such as sepsis and prolonged recovery time. A prior study in a rat model of hindlimb unloading after burn supports that bedrest contributes significantly to muscle atrophy. The aim of our study was to evaluate if exercise mitigates the loss of muscle in this animal model.

Methods:
Two groups of 24 Sprague-Dawley rats were randomly assigned to burn ambulatory (B/A), burn hindlimb unloading (B/H), sham ambulatory (S/A), or sham hindlimb unloading (S/H). One group was trained to perform twice daily weighted resistance climbing of 1 meter with 5 repetitions; the other group had no exercise. Rats received a full thickness scald burn of 40% total body surface area or sham and were allowed to ambulate or were placed in a tail traction system for hindlimb unloading. On day 14 in situ isometric forces were measured on the left soleus muscle. Statistical analysis was performed with Sigma Plot using Student’s t-test, Mann Whitney, or ANOVA with Holm-Sidak method where appropriate. 

Results:
The soleus wet weight was lower in the hindlimb (144 mg) and the exercise (136 mg) versus the ambulatory (190 mg, p<0.001), and no exercise (180 mg, p=0.01) groups. There was no difference in weights between burn and sham. Twitch was significantly lower in the hindlimb group: 31 vs 12 g (p<0.001). Compared to no exercise, the B/H exercise group had a significantly higher twitch force 14 vs. 8 g (p=0.04). Across all other factors there was no significant difference in the twitch between exercise and no exercise. There was a significantly lower tetanic force in the hindlimb group: 55 vs 148 g (p<0.001). B/A had a lower tetanic force in the exercise group versus no exercise: 118 vs 165g (p=0.02). In B/H no difference in tetanic force was seen with or without exercise. All hindlimb groups had significantly lower specific tetanic force than ambulatory: 12 vs. 22 N/cm2 (p<0.001). The specific tetanic force in B/H was significantly higher in exercise versus no exercise: 14 vs. 7 N/cm2 (p=0.008). Fatigue index was significantly lower in the ambulatory (55%) and exercise (52%) groups versus hindlimb (69%) and no exercise (73%) groups (p=0.03, p=0.002 respectively). Muscle function of all groups included in table.

Conclusion:
Hindlimb unloading is a significant factor in muscle atrophy with or without burn. Exercise increased soleus twitch and specific force in this model. However, there was a surprising decrease in muscle mass with exercise in all groups and a decrease in the fatigue index. These findings suggest that exercise contributes to a functional muscle change in a model of disuse and critical illness. 
 

4.08 Stress Conditions Modulate Adipose Tissue Inflammatory Response and Subsequent Organ Injury

Posted on December 22, 2014

D. A. Edelman1, D. M. Liberati1, L. N. Diebel1  1Wayne State University,Surgery/School Of Medicine,Detroit, MI, USA

Introduction: Obesity is an independent risk factor for ARDS and organ failure after severe trauma.  Adipose tissue (AT) composed of adipocytes, macrophages, and other immune cells is a source of pro-inflammatory mediators that are associated with a chronic low grade inflammatory state in the obese patient.  Obesity is also associated with activation of the sympathetic nervous system, which has been linked to shock induced gut and lung injury in the trauma setting.  We hypothesized that sympathomimetic stimulation of adipose tissue would augment inflammatory signaling from adipose tissue and contribute to lung injury and ARDS after trauma.

Methods: Cell co cultures of mature adipocytes and macrophages (RAW264.7) were established and then incubated with either low or high concentrations of epinephrine (10-6 or 10-3  μM respectively). Cell culture supernatants (sup) were obtained at 12 hrs, and AT derived TNF α and IL-6 determined. In separate experiments, human microvascular endothelial cell (HMVEC) monolayers were incubated with adipocyte macrophage sup and HMVEC apoptosis (%apo), ICAM expression (MFI) and FITC-dextran permeability determined.

Results: No difference was seen in co culture data between the low dose epinephrine groups and the no dose epinephrine groups (p<0.001).

Conclusion: Both adipocytes and macrophages contribute to the "obesity related" proinflammatory state. Augmentation of this response after stress related sympathetic activation could contribute to lung injury and other remote organ failure in the injured obese patient. This response seems primarily due to stimulation of the adipocyte component of adipose tissue. The clinical impact likely depends on the magnitude of injury and the distribution/percent total body fat of the patient. Modification of the stress response following trauma may be a therapeutic target in this population. 

 

4.09 Inhibition of Histone Deacetylase 6 Protects the Intestinal Tight Junctions in Hemorrhagic Shock

Posted on December 22, 2014

Z. Chang1, W. He1, B. Liu1, I. Halaweish1, T. Bambakidis1, Y. Li1, H. B. Alam1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction: We have previously shown that treatment with histone deacetylase (HDAC) 6 inhibitor Tubasatin A (Tub A) can improve outcome in a rat model of septic shock. Also, we have demonstrated that circulating levels of claudin 3, a tight junction (TJ) protein, increase after shock and return to normal after treatment with VPA (pan-HDAC inhibitor). This study investigated whether specific inhibition of HDAC 6 could protect intestinal cellular TJ and improve survival after HS.

Methods: Experiment I: Sprague Dawley rats underwent hemorrhagic shock (40% blood loss over 10 min) followed by treatment with Tub A (70 mg/kg), without any fluid resuscitation. The experimental groups were: (1) sham (no hemorrhage, no treatment), (2) control (hemorrhage, no treatment), and (3) treatment (hemorrhage with Tub A treatment). Animals were sacrificed 6 hrs later, and intestinal tissues were used to create whole cell lysate, which were analyzed for acetyl-tubulin, total tubulin, claudin 3 and zonula occludens 1 (ZO-1) proteins by Western blot. Experiment II: human intestinal epithelial cells (Caco-2) were divided into 3 groups: (1) sham (no hypoxia), (2) control (hypoxia, no treatment), (3) treatment (hypoxia, treatment with tub A). After 12 hours in a hypoxia chamber, cells were analyzed for viability, Lactate dehydrogenase (LDH) levels measuring cellular injury, and TJ protein (claudin 3 and ZO-1) levels.

Results: Hemorrhage decreased, whereas treatment with Tub A increased, expression of claudin 3 and ZO-1. Administration of Tub A also acetylated the tubulin protein. In vitro study showed that hypoxia decreased the viability of caco- 2 cells, which was prevented by Tub A treatment. Similar results were seen when cellular cytotoxicity was determined by the LDH activity. The expression of TJ protein claudin 3 and ZO-1 was significantly decreased by hypoxia, which was significantly prevented by  Tub A treatment. Immunofluorescent study further confirmed that Tub A attenuated the hypoxia-induced claudin 3 and ZO-1 signal loss in the caco-2 cells (Figure). 
 

Conclusion: Selective inhibition of HDAC6 with Tub A preserves the expression of intestinal TJ proteins in models of hemorrhagic shock in vivo and cellular hypoxia in vitro.

4.11 Opioids Promote Anastomotic Leak Following Colo-rectal Surgery in Rats: The Role of the Microbiota

Posted on December 22, 2014

B. A. Shakhsheer1, J. R. Defazio1, J. N. Luo1, R. Klabbers2, I. D. Fleming1, N. Belogortseva1, A. Zaborin1, O. Zaborina1, J. C. Alverdy1  1Pritzker School Of Medicine, University Of Chicago,Department Of Surgery,Chicago, IL, USA 2Radboud University Nijmegen Medical Centre,Department Of Surgery,Nijmegen, , Netherlands

Introduction:  The most dreaded complication after resection of the gastrointestinal tract is anastomotic leak. The effects of opioids on outcomes after gastrointestinal surgery continue to be discovered. Use of patient-controlled analgesia pumps has been associated with increased deep surgical site infection rates by unknown mechanisms. Two findings from our lab may shed light on the mechanisms by which opioids increase anastomotic leak rates: 1. opioids (morphine) directly enhance the virulence of intestinal pathogens and 2. intestinal bacteria play a key causative role in the pathogenesis of leak. Therefore the aim of this study was to examine the effect of opioids on colonic anastomotic leak in a rat model.

Methods:  Rats undergoing one-centimeter colectomy at the peritoneal reflection and primary anastomosis were treated with slow release subcutaneous morphine pellets or placebo pellets implanted in the nape of the neck. Rats were sacrificed on post-operative day six and autopsied for gross signs of anastomotic leak. Microbial composition and phenotype (i.e. collagenase production) was investigated via culture of tissues and intraperitoneal lavage. Local anastomotic tissue was subjected to high magnification microscopy and phenotype analysis.

Results: Rats treated with high-dose (15-mg sustained release) subcutaneous morphine pellets developed a 56% leak rate compared with a 3% leak rate in the placebo treated group (n=73, p=0.0045) High powered images of the anastomotic site demonstrated mucosal ulceration in the morphine group with visible disruptions in the anastomotic integrity whereas no such findings were observed in the placebo treated group. Culture of local tissue and intraperitoneal lavage fluid identified the presence of gram-negative bacteria producing high levels of collagenase which may a play role in anastomotic disruption and non-healing. 

Conclusion: Morphine significantly increases anastomotic leak rates in rats. The role of microbial composition (i.e. gram negative pathogens) and phenotype (i.e. collagenase productions) remains to be investigated and their predictive value confirmed. Taken together these findings provide a rationale to limit opioid use following colorectal surgery and/or to block their peripheral effects with selective opioid antagonists as a countermeasure to prevent deep organ space infection and anastomotic leak.

 

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