27.08 The Use of Cadaveric Human Intestine as a Source of Intestinal Stem Cells

Posted on December 22, 2014

A. Scott1, J. D. Rouch1, B. A. Kokubun1, H. A. Khalil1, N. Y. Lei1, B. Olack7, M. Lewis5, J. C. Niland8, M. G. Stelzner1,4, J. C. Dunn1,6, M. G. Martín2,3  1University Of California – Los Angeles,Division Of Pediatric Surgery, Department Of Surgery, David Geffen School Of Medicine At UCLA,Los Angeles, CA, USA 2University Of California – Los Angeles,Division Of Gastroenterology And Nutrition, Department Of Pediatrics, Mattel Children’s Hospital And The David Geffen School Of Medicine At UCLA,Los Angeles, CA, USA 3University Of California – Los Angeles,Eli And Edythe Broad Center Of Regenerative Medicine And Stem Cell Research, University Of California, Los Angeles,,Los Angeles, CA, USA 4Veterans Affairs Greater Los Angeles Healthcare System,Department Of Surgery,Los Angeles, CA, USA 5Veterans Affairs Greater Los Angeles Healthcare System,Department Of Pathology,Los Angeles, CA, USA 6University Of California – Los Angeles,Department Of Bioengineering, School Of Engineering,Los Angeles, CA, USA 7City Of Hope National Medical Center,Department Of Information Sciences · Integrated Islet Distribution Concortium & Intestinal Stem Cell Consortium – Coordinating Center,Duarte, CA, USA 8City Of Hope National Medical Center,Department Of Information Sciences, Intestinal Stem Cell Consortium,Duarte, CA, USA

Introduction: Human intestinal stem cells (hISCs) can be reliably isolated from both human intestine. Intestinal samples typically come from surgical patients. However, samples are exposed to warm ischemia, which often results in decreased hISCs quality and quantity, affecting the overall generation and growth of spheroids and enteroids. Furthermore, the use of surgical samples is limited by the case load of the providing hospital and inability to ship samples beyond local areas. The use of cadaveric intestinal samples to isolated hISCs can potentially serve as an excellent alternative to isolate hISCs.

Methods: Human small intestinal samples were obtained from surgical patients (n=6) and cadaveric donors (n=4). Portions of the cadaveric samples were sectioned off for crypt isolation at 24 hour intervals for 6 days. Both surgical and cadaveric samples underwent crypt isolation in a similar manner. The mucosal segments were washed with phosphate buffered saline and incubated at 4°C in solution for 30 minutes. Intestinal crypts were released from segments using centrifugal force, filtered and re-suspended in basic media. Intestinal crypts were suspended in Matrigel and supported with 1 of 6 types of growth media. After 1 week of growth, generated spheroids and enteroids were subcultured, processed for RNA and histology.

Results: Intestinal crypts were successfully isolated up to 144 hours post procurement. Using an isolation method developed for surgical samples, crypt isolated from cadaveric samples were significantly decreased when compared to surgical samples (2,681±1589 vs. 12,350±1520 crypts/gm, p<0.05, student t test). However, this decrease did not affect spheroid and enteroid generation and growth. Cadaveric hISCs supported with growth media containing myofibroblast conditioned media (CM) or 10nM PGE2 with and without Wnt3A CM resulted in spheroid generation and growth similar to surgical samples, up to 144 hours post procurement. Similarly, cadaveric crypts supported with growth media containing 2.5µM GSK inhibitor, jagged-1, and Wnt3A CM resulted in enteroid generation and growth similar to surgical samples. At all time points, spheroids and enteroids subcultured similarly to surgical samples. mRNA expression profile showed that generated spheroids in both cadaveric and surgical samples had increased levels of several very specific markers. Similar expression of CDX2, MUC2, and DEFA5 was seen in enteroids of both sample types.

Conclusion: Cadaveric small bowel crypts can be isolated and cultured up to 6 days post procurement. Cultured cadaveric crypts form into spheroids and enteroids and subculture similar to crypts from surgical samples. Cadaveric human intestine is an excellent source to generate spheroids and enteroids, which can be subsequently be used for research and possible clinical applications. Focus on the optimization of the crypt isolation specifically for cadaveric samples should improve crypt yield.

 

27.09 Bone Marrow Stromal Cells Remediate Radiation in Distraction Osteogenesis via Histological Evaluation

Posted on December 22, 2014

S. S. Deshpande1, A. Zheutlin1, S. Kang2, T. Patel1, A. Donneys1, P. Felice1,3, N. Noah1, J. Rodriguez1, Y. Polyatskaya1, S. R. Buchman1  1University Of Michigan,Plastic Surgery,Ann Arbor, MI, USA 2University Of Michigan,Otolaryngology,Ann Arbor, MI, USA 3Medical University Of South Carolina,General Surgery,Charleston, Sc, USA

Introduction:

Radiation is known to be detrimental to bone and soft tissue repair, resulting in an unacceptably high incidence of devastating wound healing complications. This is effected through a mechanism of both direct cellular and vascular depletion. We sought to utilize bone marrow stromal cells (BMSCs), which have previously been shown to enhance the generation of new bone during distraction osteogenesis (DO), to remediate this deficiency. The purpose of this study was to allow for the successful utility of distraction osteogenesis in an irradiated field utilizing intra-operatively placed BMSCs for the purpose of craniofacial reconstruction in head and neck cancer victims.

Methods:

29 male Lewis rats were split into three groups, DO (n=10), XRT/DO (n=10) and BMSC (n=14). XRT and DO underwent 5 day fractionated XRT of the left mandible at 7 Gy per day and were allowed to recover for two weeks. All groups underwent mandibular distractor placement. The BMSC group received a Surgifoam scaffold with 2 million BMSCs intra-operatively placed within the distraction gap. Groups were distracted at 0.3mm every 12 hrs to a 5.1mm (a critical-sized defect for an irradiated, distracted mandible), and sacrificed on post-operative day 40. Coronal sections were obtained and stained using Hematoxylin & Eosin (H&E), Safranin O, and Gomori Trichrome. Statistical analysis was performed with ANOVA and subsequent Tukey or Games-Howell post-hoc tests, dependent on data heterogeneity.

Results:

Gomori Trichrome demonstrated a significantly increased osteocyte number (87.13 + 8.08 vs 67.86 + 9.47, p=0.000) and significantly decreased empty lacunae (2.17 + .62 vs 15.64 + 7.80, p=0.000) in BMSC compared to XRT/DO. There were no significant differences between DO and BMSC. Safranin O demonstrated no cartilage presence. H&E staining demonstrated more woven bone within the regenerate of DO as well as XRT/DO/PTH specimens.

Conclusion:

The vascular environment of bone is essential for its capacity to heal. We demonstrated both qualitative and quantitative metrics of radiation-induced remediation of cellularity utilizing bone marrow stromal cells, demonstrating its potential for use in irradiated fields. BMSC therapy was able to remediate the detriments to osteocyte number as well as prevent empty lacunae formation. Furthermore, BMSCs stimulated new osteoid growth, as established by H&E. Finally, BMSCs maintains the intramembranous healing mechanism intrinsic to DO, as the Safranin O stain showed no evidence of a cartilaginous intermediate. As such, this abstract has demonstrated that intra-operatively placed BMSC therapy is a powerful tool for remediating the damage of radiation and allowing for successful utilization of distraction osteogenesis.

27.10 ORGAN-SPECIFIC DIFFERENCES IN FIBRINOLYSIS DURING ISCHEMIA

Posted on December 22, 2014

E. Gonzalez1, H. B. Moore1, M. P. Chapman1, M. Fragoso1, A. P. Morton1, C. C. Silliman1,2, A. Banerjee1, E. E. Moore1,3  1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2Bonfils Blood Center,Department Of Researcj,Denver, CO, USA 3Denver Health Medical Center,Department Of Surgery,Denver, CO, USA

Introduction:  The fibrinolytic response to trauma and hemorrhagic shock has been thus far characterized as a hyper-fibrinolytic state, which exacerbates coagulopathic bleeding. However, recently we have identified a subpopulation of patients with impaired fibrinolysis, also referred to as fibrinolysis shutdown, which favors unregulated clotting, particularly in the microvasculature. This spectrum of fibrinolysis may occur with simultaneous heterogeneity throughout tissue beds. We hypothesized that the response to ischemia is organ specific, with distinct patterns of fibrinolysis. 

Methods:  Six Yorkshire swine were anesthetized, and after laparotomy, the kidney, small bowel, liver, and spleen were subjected to controlled ischemia by arterial clamping. Venous samples were obtained from these organs at baseline (before ischemia), then at 15 and 30 min. after clamping, and 5 min. after reperfusion. A systemic sample from the saphenous vein was also obtained at theses time points. A systemic mean arterial pressure of 35 mmHg was induced after clamping during this period of time. Thrombelastography (TEG) was performed from re-calcified citrated samples between 20-40 min. from collection. Changes in TEG variables for each organ along these time-points were compared by the Friedman test for significance (p<0.05), with post-hoc pairwise comparisons (Bonferroni adjusted p-value).
 

Results: The TEG variables R-time, alpha-angle, and maximal amplitude (MA), all progressively demonstrated a coagulopathic profile with ischemia time, and did not normalize after reperfusion, for all organs and for the systemic venous samples. Fibrinolysis quantified by TEG (%LY30: percent of clot lysis 30 min. after reaching MA) progressively decreased with ischemia, demonstrating fibrinolysis shutdown; this was statistically significant for the kidney and liver (Fig. 1). However, the systemic venous samples demonstrated hyper-fibrinolysis proportional to ischemia time, which was also statistically significant. All organs had statistically significantly less fibrinolysis than the systemic venous sample at 15 min. and 30 min. of ischemia, and after reperfusion (Fig. 1).

Conclusion:  Despite evidence of systemic hyper-fibrinolysis during shock, paradoxically, fibrinolysis shutdown occurs simultaneously at an organ level, particularly in the kidney and liver. Consequently, treatment of hyperfibrinolysis with anti-fibrinolytics, such as tranexamic acid, may have adverse effects in certain organs’ microvasculature. These differences in the spectrum of fibrinolysis provide further insight into the pathophysiology of trauma-induced coagulopathy, allowing for study of its optimal diagnosis and management. 

 

28.01 Variation in Transfusion Practices and the Effect on Outcomes after General or Vascular Surgery

Posted on December 22, 2014

Z. M. Abdelsattar1, S. Hendren1, S. L. WONG1, D. A. Campbell1, P. K. Henke1  1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA

Introduction:  There are no definitive data on the efficacy of blood transfusion in improving outcomes, and recent data suggest alarming associations with harm. Postoperative transfusion practices and their effect on short-term outcomes in patients undergoing non-cardiac general and vascular surgery are not well understood.

Methods:  This is a retrospective cohort study using prospectively collected data. Demographic, operative and outcomes data for 48,720 patients undergoing general or vascular surgery at 52 hospitals between 7/2012 and 4/2014 were reviewed. Receipt of a blood transfusion within 72 hours after the operation was the main exposure variable. Thirty-day infectious complications, ischemic cardiac events, any morbidity, and mortality were the outcomes of interest. Propensity score matching across 64 different variables was used to estimate the effect of transfusion on outcomes. Hospitals were divided into restrictive, average, or liberal transfusers based on aggregate averages of nadir postoperative hematocrit values in transfused patients. Hierarchical models were used to estimate hospital level risk-adjusted outcomes.

Results: In this cohort, 2,243 (4.6%) patients received a postoperative blood transfusion. After propensity matching, a postoperative transfusion was associated with increased 30-day mortality (3.6% excess absolute risk; p=0.001), any morbidity (9.7% excess absolute risk; p<0.001), and infectious morbidity (5.4% excess absolute risk; p<0.001). However, a transfusion was associated with a 4.7% absolute risk reduction in postoperative ischemic cardiac events (p=0.001). At the hospital level there was wide variation in transfusion practices as evident by nadir hematocrit values for transfusions (Figure). Hospitals with liberal transfusion practices were twice as likely to transfuse patients and had higher risk-adjusted mortality rates than restrictive hospitals (3.1% vs. 2.2%; p<0.001)

Conclusion: Postoperative transfusions are associated with significantly increased adverse postoperative outcomes with the exception of ischemic cardiac events. Hospitals that are liberal in their transfusion practices also have markedly worse outcomes, suggesting potential interventions for quality improvement.

 

28.02 Preoperative HbA1C is a Better Predictor than Postoperative Glucose of Major Complications

Posted on December 22, 2014

C. J. Goodenough1, M. T. Nguyen1, D. H. Nguyen1, J. S. Roth2, C. J. Wray1, L. S. Kao1, M. K. Liang1  1University Of Texas Health Sciences Center At Houston,Surgery,Houston, TX, USA 2University Of Kentucky,Surgery,Lexington, KY, USA

Introduction:
Glycosylated hemoglobin(HbA1C) is diagnostic of and a measure of chronic hyperglycemia. Both HbA1c and perioperative hyperglycemia have been targeted as modifiable risk factors for post-operative complications. The aim of this study was to determine whether HbA1C or peak 24-hour post-operative peak glucose has a stronger association with major complications and to identify the peri-operative goals for both to minimize complications.  

Methods:
A prospective study of all abdominal surgeries from a single institution from 2007-2010 was performed. All patients with a HbA1C within 3-months prior to surgery and at least one 24-hour post-operative glucose were included; only the peak glucose level was used. During the study period, standardized order sets for blood glucose control were routinely utilized. Variables were collected according to criteria established by the National Surgical Quality Improvement Project. The primary outcome was major complication defined as any Dindo-Clavien 3-5 complication within 30-days of surgery. Stepwise, multivariable analysis was performed including clinically relevant variables chosen a priori. Predicted probabilities for major complications were calculated at fixed HbA1C and peak glucose levels.

Results:

Among 374(out of 1017) patients who had both a HbA1c and post-operative glucose level, 92(24.6%) experienced a major complication. One-third(n=129) of these patients had a diagnosis of diabetes mellitus. On univariate analysis, both HbA1c and peak glucose level were predictive of complications. When HbA1c and peak glucose were considered independently, HbA1c≤6% and peak glucose 80-119 mg/dL had the lowest rates of major complications. When both HbA1c and peak glucose were included in the multivariable analysis, glucose was no longer statistically significant. HbA1C>6% predicted major complications (OR 2.12, 95% CI 1.19-3.76). The predicted probabilities of major complications demonstrated that within each HbA1c stratum, there were fewer major complications at lower peak glucose levels excluding <80 mg/dL (Table). However, the higher the HbA1c, the more likely the patient was to have elevated peak glucose levels.

Conclusion:

Both HbA1c and peak glucose are predictive of major complications after abdominal surgery, although HbA1c is a better predictor. Nonetheless, both should be considered together in stratifying patients’ risks for major complications. Whether these are both modifiable risk factors depends upon the urgency of the procedure and feasibility of achieving the target levels given the patient severity of disease and compliance. Further studies are necessary to determine safe, effective methods for optimizing both acute and chronic glycemic control around surgery.

28.03 Leukopenia is an Independent Predictor of Poor Outcomes following Emergent Abdominal Surgery

Posted on December 22, 2014

B. C. Gulack1, B. R. Englum1, D. D. Lo1, D. P. Nussbaum1, J. E. Keenan1, J. E. Scarborough1, M. L. Shapiro1  1Duke University Medical Center,Department Of Surgery,Durham, NC, USA

Introduction: There is little data currently available to guide surgical decision making regarding emergent surgical interventions on leukopenic patients.  The purpose of this study was to investigate the impact of leukopenia among patients undergoing emergency abdominal operations in order to better guide preoperative patient discussions.

Methods: The 2005-2012 American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database was queried to identify patients who underwent emergent laparotomy.  Patients were stratified by pre-operative white blood cell count (4.0-12.0 vs. <4.0).  Baseline demographics, comorbidities, and outcomes were compared.  Multivariable logistic regression was performed to estimate the independent association between leukopenia and mortality taking into account the robust array of patient-related factors.

Results: 22,097 patients met inclusion criteria, 2,252 (5.5%) of whom were leukopenic prior to surgery.  There were notable differences between groups including baseline functional status, ASA class, steroid use, pre-operative chemotherapy/radiation, and pre-operative sepsis among others.  Unadjusted comparison demonstrated significantly increased organ space infections (6.8% vs 4.6%, p <0.001), post-operative pneumonia (11.9% vs. 8.4%, p <0.001), prolonged mechanical ventilation (31.4% vs. 16.3%, p <0.001), new onset post-operative dialysis requirement (2.1% vs. 1.3%, p <0.001), hospital length of stay (10 vs. 8 days, p <0.001), and 30-day mortality (27.8% vs. 12.8%, p <0.001) in the leukopenia group (Figure).  After multivariable adjustment for patient-related factors, leukopenia was maintained as a significant predictor of mortality.

Conclusion: Leukopenia is an independent predictor of mortality in patients undergoing emergent laparotomy.  Understanding the risk of complications and mortality associated with these procedures is pertinent for pre-operative patient discussions.

28.04 Be Aware of Blood Transfusion in Colorectal Surgery for Diverticular Disease

Posted on December 22, 2014

S. Tam3, P. Chung2, R. Lee2, A. Alfonso2, G. Sugiyama2  2SUNY Downstate Medical Center,Department Of Surgery,Brooklyn, NY, USA 3SUNY Downstate,College Of Medicine,Brooklyn, NY, USA

Introduction:  Diverticular disease is one of the most frequent causes of severe lower gastrointestinal bleeding and about 15% of patients admitted for acute diverticulitis will require emergent surgical treatment. In 2011, almost 14 million units of red blood cells were transfused in the United States with 20% related to surgical procedures. Previously, studies have focused on the detrimental effect of blood transfusion in colorectal cancer resection but there is a lack of data concerning blood transfusion in benign colorectal surgery. Our objective was to analyze the effect of blood transfusion on the postoperative outcomes of emergent colorectal resection for diverticular disease.

Methods:  Patients with diverticulosis or diverticulitis of the colon who underwent emergent colectomy or proctectomy from 2005-2010 were selected from the American College of Surgeons National Surgical Quality Improvement Program and separated based on blood transfusion status. Patients with a preoperative hematocrit less than 30% were identified since this is a common threshold for transfusion. Patient demographics, preoperative and intraoperative variables were analyzed. Multivariate logistic and linear regression was performed to determine the effect of blood transfusion on 30-day mortality, morbidity, and length of hospital stay.

Results: A total of 3,385 patients were included and 683 (20.2%) patients received blood transfusions. Overall, blood transfusions were associated with increased mortality (odds ratio [OR] 2.15), morbidity (OR 1.66), infectious outcomes (OR 1.36), respiratory outcomes (OR 1.77), vascular outcomes (OR 1.53) and length of stay (mean difference 2.22 days). In patients with hematocrit <30%, blood transfusion was associated with increased mortality (OR 2.90), morbidity (OR 1.77), infectious (OR 1.53) and respiratory (OR 1.75) outcomes.

Conclusion: Blood transfusion in patients with acute diverticular disease following emergent colorectal resection is associated with increased 30-day mortality, morbidity, and length of stay. Although a hematocrit of less than 30% has been described as a threshold for transfusion, blood transfusion in these patients was also associated with increased mortality and morbidity. This large study demonstrates that surgeons should be aware of the postoperative risks of blood transfusion in patients undergoing colorectal surgery for diverticular disease.

 

28.05 Surgical Outcomes of Cholecystectomy Following Cholecystostomy for Acute Calculous Cholecystitis

Posted on December 22, 2014

H. Mazeh1, I. Mizrahi1, J. Yuval1, G. Almogy1, M. Bala1, N. Abu Ata1, E. Kuchuk1, J. Rachmuth1, A. Nissan1, A. Eid1  1Hadassah-Hebrew University Medical Center,Surgery,Jerusalem, , Israel

Introduction:
The role of percutaneous cholecystostomy (PC) in the management of patients with acute calculous cholecystitis (ACC) remains controversial, and data is limited to small-scale studies. The aim of the present study is to report peri-operative outcomes of a large cohort of patients, managed with PC prior to their cholecystectomy.

Methods:
The hospital Electronic Medical Record (EMR) was searched for all patients underwent delayed cholecystectomy due to ACC, between 2003- 2012. All patients managed by PC prior to surgery were identified. Patient EMR were reviewed to record demographics, clinical presentation at index admission, and during interval period, and operative and post-operative data. Patients who underwent delayed cholecystectomy following ACC without PC during the study period, served as control group.

Results:
During the study period 640 patients underwent delayed laparoscopic cholecystectomy (LC) following ACC at our institution. Of the entire cohort, 163 (25%) patients underwent delayed LC with prior PC insertion and 477 (75%) patients underwent interval LC with no prior PC intervention. Patients in the PC group were older (64±1 vs. 48±0.8, p<0.001), had a higher ASA score (2.02 vs. 1.78, p=0.01), and had significantly more co-morbid conditions (p<0.001). Combined ACC with cholangitis at the index admission was more commonly observed in the PC group (7% vs. 1%, p<0.001). The accumulated length of stay (LOS) for all admissions was significantly longer in the PC group (16.2±0.4 days, vs. 9.7±0.1 days, p<0.001). During the time interval between ACC and LC there were  12% of accidental tube ejection, 4% of peri-tubal leakage, and 8.5% of patients requiring tube re-insertion. Emergency room referrals (30% vs. 8%, p<0.001), as well as re-admission rates (33% vs.12%, p<0.001), within 90 days post-surgery, were higher in the PC group. Laparoscopic to open conversion rate was higher in the PC group (11% vs. 3.7%, p=0.001) and operative time was significantly longer (142±4 minutes vs. 107±4 minutes, p<0.001). Patients in the PC group presented with a higher rate of biliary tract injury (9.2% vs. 2%, p<0.001) and surgical site infections (SSI), both superficial (4.9% vs. 1%, p=0.004), and deep (7.3% vs. 2.9%, p=0.04). Comorbidity related complications were similar between the groups. 

Conclusion:
Managing patients with ACC using PC is associated with longer LOS, higher rate of readmissions, and most importantly, higher conversion rate, biliary tract injuries, and SSI. 

 

24.08 Selective inhibition of Histone Deacetylase 6 Improves Survival in a Lethal Two-hit Model

Posted on December 22, 2014

X. Cheng1, Z. Liu1, B. Liu1, Y. Li1, H. B. Alam1  1University Of Michigan,General Surgery,Ann Arbor, MI, USA

Introduction: Hemmorrhagic shock (HS) followed by a subsequent infection (“second hit”) is highly lethal. We have previously demonstrated that treatment with valproic acid, a pan histone deacetylase (HDAC) inhibitor, improves survival in a rodent “two-hit” model. In the present study, our aim was to determine whether selective inhibition of histone deacetylase 6 with Tubstatin A (Tub A) could prolong survival in a 2-hit model.

Methods: C57Bl/6J mice were subjected to HS (40% blood loss) and then randomly divided into 2 groups (n=13/group): The treatment group was injected (intraperitoneal) with Tub A (70 mg/kg) dissolved in 1 µl/g of dimethyl sulfoxide (DMSO), whereas the vehicle (Veh) group was given 1 µl/g of DMSO. After 24 h, all mice were subjected to cecal ligation and puncture (CLP) followed by a second dose of Tub A or DMSO. Survival was monitored for 10 days. In a parallel study, peritoneal irrigation fluid and liver tissues were collected at 3h after CLP.  Enzyme-linked immunosorbent assay was performed to determine the myeloperoxidase (MPO) activity (marker of  neutrophil activation) and TNF-α and IL-6 concentrations in the peritoneal irrigation fluid, whereas real-time PCR was performed to measure the relative mRNA levels of TNF-α and IL-6 in the liver tissue.

Results: Tub A administration significantly improved survival compared to the control (69.2% vs 15.4%, p<0.0074) (Figure). In addition, Tub A significantly suppressed MPO activity (169.9±8.4 ng/ml vs 70.4±17.4ng/ml; p=0.0001), and inhibited levels of cytokine TNF-α and IL-6 in the peritoneal fluid (TNF-α: 105.7± 4.7 pg/ml vs 7.4±2.4 pg/ml; IL-6: 907.4±2.3 pg/ml vs 483.6 ±1.6 pg/ml; p=0.0001) compared to the vehicle control. Meanwhile, the hepatic gene expression of these cytokines was significantly lower (TNF-α: 18.9±7.8 vs 0.92±0.25; IL-6: 43.3±5.8 vs 8.3±3.4; p=0.0001) in the Tub A treated animals.

Conclusion: Tub A treatment significantly improves survival, attenuates inflammation and down regulates the gene expression and protein levels of TNF-α and IL-6 in a rodent two-hit model.

 

24.09 Selective Inhibition of SIRT2 improves Outcomes in a Lethal Septic Model

Posted on December 22, 2014

T. Zhao1, Y. Li1, B. Liu1, R. T. Bronson2, H. B. Alam1  2Harvard School Of Medicine,Brookline, MA, USA 1University Of Michigan,General Surgery,Ann Arbor, MI, USA

Introduction: Six isoforms of histone deacetylase (HDAC) Class III have been reported- Sirtuin (SIRT) 1, 2, 3, 4, 5, and 6. We recently demonstrated that EX-527, an inhibitor of SIRT1, improves survival in a lethal cecal ligation and puncture (CLP) model. The aim of this study was to determine if selective inhibition of SIRT2 with AGK2 could also improve survival in a lethal septic model, and attenuate the inflammatory response.

Methods: Experiment I: C57BL/6J mice were intraperitoneally injected with either AGK2 (82 mg/kg) dissolved in dimethyl sulfoxide (DMSO) or DMSO only, and 2 h later subjected to CLP (n=9/group). Survival was monitored for 10 days. Experiment II: animals, treated the same way as Experiment I, were grouped into (i) DMSO vehicle, and (ii) AGK2 (n=10-14/group), with sham-operated animals serving as controls. Peritoneal fluid and blood samples were collected for measurement of cytokines at 24 or 48 h. Blood at 48 h was also used to assess the coagulation status using Thrombelastography (TEG). In addition, long bones (femurs and tibias) were harvested (n=5-6/group) at 48 h to determine morphological changes in the bone marrow by H&E staining. Bone marrow atrophy was quantified by a blinded pathologist. Experiment III: Normal primary splenocytes were cultured, and treated with lipopolysaccharide (LPS) in the presence or absence of AGK2 (10 μM) for 6 h to assess cytokine production (n=4/group).

Results: AGK2 significantly improved survival (figure), and attenuated the levels of cytokines in the circulation (TNF-α: 298.3±24.6 vs. 26.8±2.8 pg/ml, p=0.003; IL-6: 633.4±82.8 vs. 232.6±133 pg/ml, p=0.034) and peritoneal fluid (IL-6: 704.8±67.7 vs. 391.4±98.5 pg/ml, p=0.033) compared to the vehicle control. It also decreased the TNF-α and IL-6 production by the splenocytes in-vitro (TNF-α: 68.1±6.4 vs. 23.9±2.8 pg/ml, p=0.001; IL-6: 73.1±4.2 vs. 49.6±3.0 pg/ml; p=0.005). The TEG data showed that animals subjected to CLP displayed markers of coagulopathy: prolonged fibrin formation and fibrin cross-linkage time, slower clot formation, decreased platelet function and clot rigidity. Inhibition of SIRT2 was associated with dramatic improvements in fibrin cross-linkage, platelet function and clot rigidity, but without a significant impact on the clot initiation parameters. Meanwhile, inhibition of SIRT2 significantly attenuated the bone marrow atrophy (58.3±6.5 vs. 30.0±8.2%, p=0.026).

Conclusions: Selective inhibition of SIRT2 significantly improves survival, attenuates “cytokine storm” and sepsis-associated coagulopathy, and decreases bone marrow atrophy in a lethal septic model.

 

24.10 Dietary Supplementation of the AHR Ligand Indole-3-Carbonyl Reduces C. Difficile Disease in Mice

Posted on December 22, 2014

W. A. Julliard1, J. Fechner1, T. De Wolfe3, N. Safdar2, J. Mezrich1  1University Of Wisconsin,Department Of Surgery, Division Of Transplant Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Medicine, Division Of Infectious Diseases,Madison, WI, USA 3University Of Wisconsin,Department Of Food Science,Madison, WI, USA

Introduction: Clostridium difficile infection is a major epidemic in the United States responsible for over 14,000 deaths a year and greater than $1 billion a year in healthcare costs. The relationship between gut immunity and C. difficile infection is poorly understood. The aryl hydrocarbon receptor (AHR) is a receptor for endogenous and exogenous ligands, and is important in maintenance of gut immunity, generation of FoxP3+ Tregs, and the protective cytokine IL-22. AHR ligands are found in cruciferous vegetables and are made by normal bacteria and probiotics, and may be reduced after certain antibiotic treatment. We hypothesize that dietary administration of the AHR ligand indole-3-carbonyl (I3C) will alter the host immune system and protect against C. difficile infection.

Methods: Male B6 mice were placed on one of three diets for two weeks; standard mouse chow (“standard”), a diet deficient of all AHR ligands (“base diet”), and the same base diet supplemented with 0.1% I3C (“I3C diet”). After two weeks on diet, mice were started on a C. difficile model consisting of antibiotic administration followed by C. difficile inoculation. Mice were then monitored for signs of disease and overall survival. At various time points throughout the experiment, tissue was collected from the cecum to be analyzed by qRT-PCR and histopathology.

Results: Pre-inoculation, mice fed the I3C diet had extremely high levels of mRNA expression of the AHR dependent enzyme Cyp1A1 in their cecum compared to mice on the base diet. I3C mice also had higher levels of FoxP3 mRNA in their cecum compared to base diet mice. After inoculation with C. difficile, standard chow mice had a typical disease response with severe weight loss and 50% mortality by day three. Base diet mice had delayed disease onset, but by day four demonstrated severe disease and overall 87.5% mortality. In stark contrast, I3C diet mice had minimal weight loss and an overall mortality of only 25%. Post-inoculation analysis demonstrated high levels of cecal Cyp1A1, FoxP3, and IL-22 mRNA expression in I3C mice compared to base diet mice. Furthermore, histology of base diet mice demonstrated increased levels of neutrophil margination, tissue edema, and inflammation compared to I3C mice.

Conclusions: Our study demonstrates that the dietary supplementation of the AHR ligand I3C significantly reduces C. difficile associated disease in a well-accepted mouse model. The mechanism for disease prevention appears to be through activation of the AHR as indicated by elevated levels of Cyp1A1 in I3C diet mice. These results suggest a potential therapeutic option for preventing C. difficile infection and could represent a major breakthrough in C. difficile treatment.

25.01 Carbon Monoxide Protects Against Mitochondrial Injury Following Hemorrhage and Resuscitation

Posted on December 22, 2014

P. Waltz1, J. Luclano1, S. Shiva1, B. Zuckerbraun1,2  1University Of Pittsburg,Pittsburgh, PA, USA 2VA Pittsburgh Healthcare System,Pittsburgh, PA, USA

Introduction:  Currently, there is no effective resuscitative adjunct to fluid and blood products to limit tissue injury for traumatic hemorrhagic shock.  The objective of this study was to investigate the role of inhaled carbon monoxide (CO) to limit inflammation and tissue injury, and specifically mitochondrial damage, in experimental models of hemorrhage and resuscitation.

Methods:  Mice underwent hemorrhagic shock and resuscitation to a mean arterial pressure (MAP) of 20 or 25 mmHg.  Mice were resuscitated with Lactated Ringers (2:1 volume of maximal shed blood ) after  90 or 120 minutes of hypotension.  Pigs (30-35 kg) were anesthetized and bled to a MAP of 30-40 mmHg for 90 minutes, followed by initial resuscitation with Hextend (1:1 volume of shed blood).  Pigs had ongoing resuscitation and support for up to four hours.  Data were collected continuously.  Primary mouse hepatocytes were used for in vitro studies and murine skeletal muscle was used for ex vivo studies.  ANOVA was used for statistical analysis and significance was assumed with a P<0.05.

Results:Inhaled CO (250 ppm for 30 minutes) protected against mortality in severe murine hemorrhagic shock and resuscitation (HS/R) (20% vs. 80%; P<0.01).  Additionally, CO limited the development of shock as determined by arterial blood pH (7.25±0.06 vs. 7.05±0.05; P<0.05), lactate levels (7.2±5.1 vs 13.3±6.0; P<0.05), and base deficit (13±3.0 vs 24±3.1; P<0.05).  A dose response of CO (25-500 ppm) demonstrated protection against HS/R lung and liver injury as determined by MPO activity and serum ALT, respectively.  CO limited HS/R-induced increases in serum tumor necrosis factor-α and interleukin-6 levels as determined by ELISA (P<0.05 for doses of 100-500ppm).  Furthermore, inhaled CO limited HS/R induced oxidative stress as determined by hepatic oxidized glutathione:reduced glutathione levels and lipid peroxidation.  In porcine HS/R, CO did not influence hemodynamics. However,  CO limited HS/R-induced skeletal muscle and platelet mitochondrial injury as determined by respiratory control ratio (muscle) and ATP-linked respiration and mitochondrial reserve capacity (platelets).  Furthermore, in vitro and ex vivo, CO limited oxygen consumption to increase oxygen levels under hypoxic conditions, and also limited loss of mtiochondrial membrane potential, oxidative stress, and loss of ATP.

Conclusion:  CO therapy limits injury to the metabolic machinery of cells in the setting of HS/R in multiple animal models. These preclinical studies suggest that inhaled CO can be a protective therapy in HS/R, however, further clinical studies are warranted. 

25.02 Genetic Modulation to Improve Traumatic Brain Injury Induced Inflammation

Posted on December 22, 2014

J. Lee1, K. Sail1, T. Costantini1, B. Eliceiri1, R. Coimbra1, V. Bansal1  1University Of California – San Diego,Department Of Surgery, Division Of Trauma, Surgical Critical Care And Burns,San Diego, CA, USA

Introduction: The invasion of leukocytes following traumatic brain injury (TBI) may have significant consequences. Excessive leukocytes are not beneficial and instead may cause an outsized inflammatory response initiating secondary neuronal injury and exacerbating long-term cognitive dysfunction.  Therefore, modulating leukocyte migration may lead to novel therapy.  Previously, we have shown that Esophageal Cancer Related Gene 4 (Ecrg4) is a novel leukocyte chemoattractant in glioblastoma. We hypothesize that Ecrg4 is a potent mediator of TBI induced leukocyte recruitment.

Methods: A controlled cortical impact model was used to create moderate TBI in 12 wk old wild type (WT) or Ecrg4 knock-out (KO) mice. Three days post-TBI, cells were isolated and flow cytometry analysis was used gating for microglia (CD45lowCD11b+ cells), monocytes (CD45highCD11b+ cells), and lymphocytes (CD45highCD11b- cells). Brain sections were immunostained with Iba-1 and CD11b to assess recruitment and activation of microglia and migrating leukocytes. Brain homogenates were collected following TBI and cytokine levels (IL-12p70, TNF, IFN-γ , MCP-1, IL-10, and IL-6) were measured by multiplex enzyme-linked immunosorbent assay (ELISA).

Results: TBI resulted in a rapid invasion of activated microglia and inflammatory monocytes, peaking at post-TBI Day 3 and persisting at Day 7 (19 ±30 vs. 1018 ± 694; p<0.05). Invading cells expressed surface markers for activation (MHCII+ and CD86+). Ecrg4 KO mice demonstrated reduced infiltration of microglia (101 ±694 vs. 299 ±143; p<0.05) and inflammatory monocytes (314 ±155 vs. 163 ±118; p<0.05) compared with Ecrg4 WT after TBI. Activated microglia (471 ±326 vs. 125 ±63; p<0.05) were also reduced in Ecrg4 KO mice when compared to Ecrg4 WT mice.  TBI induced an increase in pro-inflammatory mediators (TNF, MCP-1, and IL-6), peaking at six hours post-TBI.  TNF levels in the brain were significantly decreased in Ecrg4 KO mice compared with Ecrg4 WT mice (31± 12 vs. 4±2pg/ml; p<0.05) following TBI.

Conclusion: Genetically deficient Ecrg4 mice had altered leukocyte invasion and microglial activation following TBI, confirming Ecrg4’s role as a novel chemoattractant. Future studies will assess whether Ecrg4-mediated leukocyte invasion alters post-TBI histopathology and neurocognitive recovery.

 

25.03 DPR Reduces Visceral Ischemia and Inflammatory Cytokines Following Hemorrhagic Shock

Posted on December 22, 2014

J. L. Weaver1, S. Matheson1, P. Matheson1, C. Downard1, R. N. Garrison1, J. Smith1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction: Hemorrhagic shock (HS) is a significant cause of morbidity and mortality in trauma patients. Treatment has traditionally been intravenous fluids and blood products to restore intravascular volume. However, studies have shown that even after blood pressure normalizes, the visceral organs remain ischemic. This can lead to organ dysfunction, failure, and death.  This prolonged ischemia is thought to be due in part to the activation of an inflammatory cascade of cytokines. Direct peritoneal resuscitation (DPR) improves blood flow to the abdominal organs. In this study we examine the effect of DPR on the body’s inflammatory response to hypotensive shock.

Methods: Rats were randomly assigned to three groups (n=8/group): 1) sham (no HS); 2) HS plus conventional resuscitation (CR); or 3) HS+CR+DPR.  Rats were hemorrhaged to 40% MAP and resuscitated with shed blood and two volumes of normal saline. Group 3 rats were given intraperitoneal injections of 30cc 2.5% peritoneal dialysis solution. Effective hepatic blood flow (EHBF) was measured using a steady state of galactose. Tissue was collected at 4 hours post-resuscitation.  Serum cytokines were measured using the Luminex immunoassay or ELISA.

Results: Central hemodynamics were restored post-hemorrhage in both groups, but EHBF fell after resuscitation was complete. This was prevented by adding DPR. The DPR groups had lower serum levels of hyaluronic acid (HA) and high-mobility group box-1 protein (HMGB-1), which are markers of cellular injury, when compared to the CR group. HMGB-1 is usually sequestered in the cell’s nucleus and immunohistochemistry staining shows this is maintained in the DPR group, but the nucleus is disrupted and HMGB-1 migrated to the cytosol in the CR group. The DPR group showed lower levels of inflammatory cytokines such as IL-1a, IL-1B, IL-6 and INF-γ. Histologic sections of terminal ileum in the DPR rats also demonstrated better preservation of architecture with less edema and necrosis.

Conclusion: The addition of DPR after HS improved visceral blood flow, demonstrated by the EHBF data. This led to a preservation of cell structure, seen on histology and via a reduction in HA and HMGB-1, which are released in cell necrosis. HMGB-1 is normally stored in the nucleus during apoptosis to prevent inflammatory signaling. It was released to the cytosol in the CR group but not the DPR group, which further proves worse necrosis with CR. DPR also returned levels of inflammatory cytokines to near-normal when compared to the CR group. These findings suggest that DPR reduces the inflammatory response which contributes to multiple organ-system failure after hemorrhagic shock, and has the potential to improve outcomes in trauma patients.

25.04 Age-Dependent Changes of Metabolic Pathways in the Lung Following Murine Hemorrhagic Shock

Posted on December 22, 2014

L. R. Klingbeil1,2, G. Piraino1, P. W. Hake1, J. R. Ledford1, B. Zingarelli1  1Cincinnati Children’s Hospital Medical Center,Department Of Critical Care,Cincinnati, OH, USA 2University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA

Introduction:  Despite advances in the management of trauma victims, mortality from hemorrhagic shock and the ensuing multiple organ dysfunction syndrome (MODS), remains high. Pediatric patients have a lower incidence of MODS, including acute lung injury, than adult patients. The molecular mechanisms underlying this age-dependent susceptibility to MODS are not fully understood. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which controls autophagy and metabolic recovery through mitochondrial biogenesis. Following activation by phosphorylation, AMPK regulates mitochondrial biogenesis via peroxisome proliferator-activated receptor γ co-activator α (PGC1-α), which can also be regulated by sirtuin 1(Sirt-1). We hypothesize that these metabolic signaling pathways are altered in the lung during hemorrhagic shock and are age-dependent.

Methods:  Hemorrhagic shock was induced in anesthetized young male (2-4 months old) and mature mice (9-10 months old) by withdrawing blood from the femoral artery to a mean arterial pressure (MAP) of 30 mmHg for 90 minutes. The mice were then resuscitated with the shed blood plus two times that amount in Lactated Ringer’s solution. Mice were sacrificed 3 hours after resuscitation and lungs were harvested for biochemical assays. Sham mice underwent a similar surgical preparation but were not bled.

Results: After hemorrhagic shock, there was marked neutrophil infiltration, as evaluated by myeloperoxidase (MPO) assay, in the lung of young mice when compared to the sham mice (201.3±19.4 versus 132.9±17.7 U/100mg tissue, p<0.05) indicating increased inflammation after hemorrhage. Mature mice also exhibited higher neutrophil infiltration in the lung after hemorrhagic shock when compared to mature sham mice (222.1±30.1 versus 167.6±12.9 U/100mg tissue, p<0.05). At western blot analysis, there was a significant increase of nuclear pAMPK (1.7±0.06 relative intensity) in lung of young mice after hemorrhagic shock when compared to age-matched sham mice (1.0±0.04 relative intensity, p<0.05) and was associated with maintenance of normal levels of PGC1-α and Sirt-1, thus suggesting the capability to mount a metabolic response. Interestingly, nuclear levels of pAMPK were significantly increased in sham mature mice (2.2±0.01 relative intensity) when compared with young sham animals (p<0.05). However, after hemorrhagic shock in lung of mature mice, there was a significant decrease in pAMPK expression (1.2±0.21 relative intensity), which was associated with a marked down-regulation of PGC1-α and Sirt-1 when compared to sham age-matched mice, thus suggesting the impairment to promote a metabolic recovery after stress.

Conclusion: Our data suggests that during hemorrhagic shock, metabolic repair mechanisms are activated in the lung and involve AMPK-dependent pathways. However, this restorative process diminishes in old age.

25.05 IL-17A/F is Associated with the Immunopathology of Trauma in Humans and Mice

Posted on December 22, 2014

M. H. Ramadan1, R. Namas1, Y. Vodovotz1, T. R. Billiar1  1University Of Pittsburg,Surgery,Pittsburgh, PA, USA

Introduction:

Excessive and sustained inflammation is associated with persistent critical illness and complications such as organ dysfunction and nosocomial infection in blunt trauma patients.  The roles of many proximal mediators of trauma induced inflammation, such as IL-6 and MCP-1 are well studied.  However, the involvement of key downstream mediators including IL-17/F remains unknown.  IL-17A/F, which can be produced by Th17 cells or certain populations of innate lymphoid cells, can promote sustained inflammation and organ injury in both acute and chronic conditions.  Here, we hypothesized that IL-17A/F levels would correlate with a complicated course in human trauma patients and organ injury in mice subjected to hemorrhagic shock and trauma (HS/T).

Methods:

Animal experiments: Anesthetized C57BL/6 mice were subjected to bilateral lower extremity injury and pressure-targeted sever hemorrhagic shock (HS) with mean BP 25-30mmHg, monitored for 2 hours via bilateral femoral cannulation. Animals were then resuscitated with X3 volume of shed blood with lactated Ringer’s solution. One group of mice received IL-17-blocking antibody 12 hours prior to HS and re-dosed at the time of resuscitation. Equal volume of PBS was administered to the mice in the control group. Plasma IL-6, MCP-1 levels and ALT were measured at 6 hours (n=6/group).

Human Samples: Plasma IL-17A/F levels were measured in the serum of 88 consented, severely injured (average ISS=26, 44 patients with a complicated course and 44 with an uncomplicated course) blunt trauma patients upon admission and over time. The two cohorts were matched for ISS, age and sex.

Results:

The human data showed elevated IL-17A/F Levels in the complicated patient group; those levels were elevated within the first 24 hours of injury. These levels remained elevated and significantly higher than the uncomplicated patient group even prior to the development of infectious complications.

In the mice we were able to demonstrate significantly elevated plasma IL-17A/F levels that peaked at 6 hours after trauma correlating with peak organ damage. There were no baseline differences between the antibody treated mice and the controls. Both the treatment and control groups had similar plasma IL-6 and MCP-1 levels indicating and equivalent proximal inflammatory responses. The Blocking of IL-17 lead to a significant reduction in liver damage, measured by 62% lower ALT p= 0.007 in the antibody treatment group; supporting our hypothesis.

Conclusion:

Our clinical data show that IL-17A/F levels correlate with persistent critical illness in human trauma patients while our mechanistic studies in a mouse polytrauma model confirms that IL-17A participates in organ damage as a mediator downstream of IL-6.  These data also suggest that IL-17 producing lymphocyte populations become activated early in clinical and experimental trauma.

25.06 Fibrinolysis Shutdown Phenotype Masks Changes in Rodent Coagulation Measured by Thrombelastography

Posted on December 22, 2014

H. B. Moore1, P. Lawson1, M. Fragoso1, E. Gonzalez1, M. P. Chapman1, F. Gamboni1, S. Mitra1, C. A. Anderson1, A. Banerjee1, C. C. Silliman1, E. E. Moore1  1University Of Colorado,Surgery,Denver, COLORADO, USA

Introduction: The use of thrombelastography(TEG) in trauma has identified unique phenotypes of coagulopathy. Pathologic hyperfibrinolysis (excessive blood clot degradation) and fibrinolysis shutdown (impaired blood clot degradation) are associated with increased mortality compared to a low level of physiologic fibrinolysis. Investigating the regulation of fibrinolysis with animal models has been challenging and has low yield to clinical translation. We hypothesize that rats have a baseline fibrinolysis shutdown phenotype and require an exogenous challenge of a profibrinolytic to differentiate mechanisms that promote or inhibit fibrinolysis. 

 

Methods: Fibrinolysis resistance was assessed by TEG with exogenous tissue plasminogen activator(tPA) titrations in rat whole blood. The experimental groups(n=9 per group) were: 1)tissue injury (laparotomy/bowel crush), 2) shock(hemorrhage to MAP < 25 mmHG), and 3) sham(arterial canulation and tracheostomy).  Blood was sampled at 30 min, and assayed with native TEG challenged with taurcholic acid(TUCA, a known inhibitor of anti-fibrinolytic protein).  The percent of remaining clot strength 30 min after reaching maximum amplitude(CL30) was used to measure clot resistance to fibrinolysis.  Plasma was assayed for tPA concentration.  Liver histology was assessed for organ damage due to oxygen sensitivity and receiving direct portal blood supply.

 

Results: Rat blood was resistant to exogenous tPA, CL30 at 150ng/ml(p=0.511) and 300ng/ml(p=0.931) was similar to baseline, while 600ng/ml(p=0.046) provoked fibrinolysis.  Baseline(p=0.679) and post-procedure CL30(p=0.505) were not significantly different between groups when using a native TEG.  TEG TUCA challenge showed no differences at baseline CL30(p=0.937); whereas, post-procedure there were differences in CL30 (Fig 1, p<0.001). This corresponded to similar plasma tPA concentrations(p=0.423) at baseline between rats and different tPA concentration(p=0.003) post procedure. The percent change in CL30 from baseline was increased in tissue injury compared to sham(p=0.048.); whereas, CL30 decreased in shock versus sham(p=0.048).  Total tPA concentration was higher in the shock group compared to trauma(p=0.009) and sham(p=0.012). Histologic evidence of cellular damage was pronounced in trauma and shock compared to baseline. 


 

Conclusion:  The TEG TUCA challenge is capable of differentiating changes in clot stability with rats undergoing diverse stresses. Tissue injury inhibits fibrinolysis while shock promotes tPA mediated fibrinolysis, consistent with our recent clinical studies.  This technical modification of TEG may be valuable for elucidating the mechanisms responsible for the spectrum of fibrinolysis observed following severe trauma.

25.07 Regulation of Hypoxic Alveolar Epithelial Cells in Lung Contusion

Posted on December 22, 2014

M. Sherman1, M. V. Suresh1, D. Machado-Aranda1, B. Thomas1, N. Talarico1, Y. Shah2, K. Raghavendran1  1University Of Michigan,Acute Care Surgery/General Surgery,Ann Arbor, MI, USA 2University Of Michigan,Molecular Biology And Integrative Physiology,Ann Arbor, MI, USA

Introduction: Lung contusion (LC) due to major thoracic trauma is a significant risk factor for the development of Acute Respiratory Distress Syndrome. The main pathological consequence of LC is hypoxia. The key mediator of adaptation to hypoxia is hypoxia-inducible factor (HIF)-1. We have recently published that a key driver of acute inflammation following LC is HIF-1α. This is based on significant reductions in lung injury and inflammation observed in type II alveolar epithelial cells (AEC) of HIF-1α conditional knockout mice. To better understand the regulation of hypoxic AEC, the current project evaluated the fate of hypoxic AEC following LC.

Methods: LC was induced using a cortical contusion impactor in WT C57Bl6 and ODD-Luc (oxygen dependent domain of HIF linked to luciferase) mice. In the WT mice, global hypoxia was confirmed by pimonidazole staining after LC. In the ODD-Luc mice, subsequent IVIS imaging was performed to assess hypoxia. To evaluate the fate of hypoxic AEC (high luciferase activity), dual staining with caspase-3, TUNEL staining (apoptosis), and Ki67 (proliferation marker) were performed. Finally, laser capture microdissection (LCM) was used to isolate hypoxic and normoxic cells after LC at 24 h, yielding adequate quantities of RNA that were subjected to real-time PCR.

Results: Using Hypoxyprobe (pimonidazole), the presence of global hypoxia with LC was confirmed in the WT mice. Lung, liver, and spleen samples ODD-Luc mice showed increased luminescence at all-time points after LC. Presence of hypoxia in AEC was not uniform.  Most of the AEC that were hypoxic following LC showed evidence of caspase-3 activation. Similar results were observed with TUNEL staining. Also, the hypoxic AEC did not show a significant increase in proliferation (ki67). Using directly conjugated fluorescent-tagged luciferase antibody, hypoxic cells were visualized and isolated by LCM. Figure 1 shows the representative immuno-fluorescent staining for luciferase positive hypoxic cells (red circles, Luc+) and the adjacent normoxic cells (blue circles, Luc-) in lung samples in ODD-Luc mice 24 h after LC as well as before and after LCM. Isolated RNA also showed significant increases in VEGFa and caspase-1 in hypoxic AEC compared to normoxic AEC.

Conclusion: Hypoxic AEC following LC specifically undergo apoptosis. Moreover, the hypoxic AEC have increased VEGFa and caspase-1 activation, which confirms our previous finding that HIF-1α regulation of AEC is likely mediated through inflammasome activation.

 

25.08 Hydrogen Inhalation Protects Against Acute Lung Injury Induced by Hemorrhagic Shock/ Resuscitation

Posted on December 22, 2014

K. KOHAMA1, H. Yamashita1, M. A. Ishikawa1, T. Nishimura1, N. Fujisaki1, J. Kotani1, A. Nakao1  1Hyogo College Of Medicine,Department Of Emergency, Disaster And Critical Care Medicine,Nishinomiya, HYOGO, Japan

Introduction: Hemorrhagic shock followed by fluid resuscitation (HS/R) triggers an inflammatory response characterized by upregulation of proinflammatory cytokines and adhesion molecules and induces pulmonary inflammation that leads to acute lung injury (ALI). Hydrogen, a novel therapeutic gas, has potent cytoprotective, anti-inflammatory, and antioxidant effects. This study aimed to examine the effects of inhaled hydrogen gas on lung injury caused by HS/R.

Methods: Sprague-Dawley rats were subjected to hemorrhagic shock by withdrawing blood to achieve a mean arterial pressure of 30±5 mm Hg for 60 minutes followed by resuscitation with shed blood and saline, as needed, to restore blood pressure. After HS/R, the rats were either maintained in room air or were exposed to 1.3% hydrogen (1.3% H2, 21% O2, 77.7% N2) in a gas-exposure chamber. Rats were randomly assigned to three experimental groups consisted of sham, HS/R (air) and HS/R (hydrogen).

Results: HS/R induced ALI, as demonstrated by significantly impaired gas exchange as well as congestion, edema, cellular infiltration, and hemorrhage in the lungs. Hydrogen inhalation mitigated lung injury, as indicated by significantly improved gas exchange 3 hours after resuscitation, and reduced congestion, edema, cellular infiltration, and hemorrhage. Hydrogen inhalation did not affect hemodynamic status or tissue oxygenation during HS/R. HS/R causes the rapid production and release of several proinflammatory mediators and induces epithelial apoptosis associated with neutrophil sequestration in the lung. Exposure to 1.3% hydrogen significantly attenuated the upregulation of the mRNAs for several proinflammatory mediators (tumor necrosis factor (TNF)-α, interleukin-6 and intercellular adhesion molecule (ICAM)-1) and reduced epithelial apoptosis. Lipid peroxidation, determined by measurement of tissue malondialdehyde (MDA) levels, was significantly reduced in the presence of hydrogen, indicating antioxidant effects.

Conclusions: Hydrogen, administered through inhalation, may exert potent therapeutic effects against ALI induced by HS/R and attenuate activation of inflammatory cascades.

25.09 The alpha-7 nicotinic acetylcholine receptor mediates the vagal anti-inflammatory response to injury

Posted on December 22, 2014

S. Langness1, B. P. Eliceiri1, V. Bansal1, R. Coimbra1, T. W. Costantini1  1University Of California – San Diego,Division Of Trauma, Surgical Critical Care, Burns, And Acute Care Surgery,San Diego, CA, USA

Introduction:  Acute lung injury is a frequent complication of severe burns and is responsible for approximately 10-15% of burn-related deaths. Acute lung injury occurs as a result of an unrestrained immune response after injury and is understood to be initiated by gut barrier failure and gut inflammation. We have previously shown that vagus nerve signaling (VNS) limits burn-induced acute lung injury through its ability to prevent gut barrier failure. While the alpha-7 nicotinic acetylcholine receptor (alpha-7 nAChR) is required to mediate the systemic anti-inflammatory effects of VNS in the spleen, the role of the alpha-7 nAChR in limiting acute lung injury via the gut-lung axis is unknown. We hypothesized that the alpha-7 nAChR is required for vagal-mediated lung protection, where the anti-inflammatory effects of VNS would be lost in alpha-7 nAChR KO mice.

Methods:  Male alpha-7 nAChR knock-out (KO) and matched control C57BL/6 wild-type (WT) mice were subjected to 30% total body surface area cutaneous burn. A separate cohort of WT and KO animals was treated with cervical VNS following injury.  Changes in gut architecture were characterized using histology.  Lung injury was evaluated by histology and by measuring changes in inflammatory cell infiltration using florescence-labeled antibodies to myeloperoxidase (MPO) and CD68, surrogate markers for neutrophils and macrophages, respectively.

Results:  Severe burn caused histologic gut and lung injury in both alpha-7 nAchR WT and KO mice.  Burn-induced lung injury was characterized by increased staining for MPO and CD68 compared to sham.  VNS prevented burn-induced gut barrier injury and lung inflammation in alpha-7 nAchR WT mice. The protective effects of VNS on gut barrier failure after injury were lost in alpha-7 nAchR KO mice. VNS also failed to prevent acute lung injury in burn-injured alpha-7 nAchR KO mice, with lung histology similar to alpha-7 nAchR WT and KO mice exposed to burn alone.  The ability of VNS to attenuate the mobilization of inflammatory cells to the lung after burn injury was also lost in alpha-7 nAchR KO mice, with MPO and CD68 staining intensity comparable to burn. 

Conclusion:  The alpha-7 nAChR is required for vagal-mediated protection against burn-induced acute lung injury.  Therapies that alter the inflammatory response after injury by either enhancing vagal activity or activating the alpha-7 nAChR may limit acute lung injury and improve outcomes in severely burned patients.

 

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