88.18 Developing Ex-Vivo Model of Left Ventricular Outflow Tract to Understand Disease Etiology?

Posted on January 31, 2019

K. Brown1, Y. Ning2, M. Kang1, M. Farenholtz2, J. Grande-Allen1, S. Keswani1,2  1Rice University,Bioengineering,Houston, TX, USA 2Texas Children’s Hospital,Department Of Surgery,Houston, TX, USA

Introduction: Turbulent flow in the Left Ventricular Outflow Tract (LVOT) is considered the main cause of Discrete Subaortic Stenosis (DSS). DSS is a heart disease characterized by the formation of a fibrotic membrane encircling the LVOT. Our lab is using DSS patient echocardiographic data to mimic pathologic conditions ex-vivo in a parallel plate flow loop bioreactor. We aim to understand the mechanism of DSS by studying cellular behavior under varying flow conditions. To understand cellular behavior, we must have an appropriate system for cells to adhere to. In this study, we compare the efficiency of cell adherence to different matrices when exposed to flow as modeled on patient echo data.

Methods:  To compare the efficiency of cell adherence with different matrices, we created gelatin, gelatin methacryloyl (gelMA), and fibronectin coatings on functionalized glass slides. Organic residues were removed from glass slides with sulfuric acid. The slides were then treated with 3-(Trimethoxysilyl)propyl methacrylate to functionalize the surface. Endocardial endothelial cells (EECs) were isolated from porcine left ventricle tissue. We then plated 1×10^6 of EECs on each slide and subjected them to flow rates observed in the LVOT.  We analyzed cell adherence using a computational algorithm to assess the efficiency of each matrix with an image analysis pipeline to assess cell morphology and cell counting. CD31 IHC is used to validate endothelial phenotype. 

Results: We created a bioreactor that could mimic flows at high, low, and static conditions. EECs were confirmed to be near 100% endothelial lineage by CD31 and DAPI staining. Cells were seeded onto gelatin, gelMA, and fibronectin matrices. Gelatin was observed to have 100% adherence at static conditions, 80% adherence at low shear, and 75% adherence at high shear. Cells under high shear flow on gelMA matrix showed a dissociation of the CD31 from the cell membrane in response to high shear as compared to low shear.

Conclusion: Cells adhered to gelatin with optimal adherence under high flow. This gel composition in the bioreactor allows us to the investigate flow disturbances in the LVOT. Interestingly, CD31 is suggested to be affected by differential shear forces and may have a role in mechanotransduction in LVOT pathology. We hope this project will open new avenues for studying DSS and other heart diseases influenced by turbulent flow.

86.10 The extent of scar formation is characterized by changes in keratinocyte proliferation

Posted on January 31, 2019

H. Li1, S. Balaji1, X. Wang1, M. Rae1, M. Chandramouli1, A. Blum1, M. Kodali1, P. Bollyky2, M. Butte3, S. Keswani1  1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Stanford University,Infectious Diseases And Microbiology And Immunology,Palo Alto, CA, USA 3University Of California – Los Angeles,Pediatrics,Los Angeles, CA, USA

Introduction: The normal course of wound healing in adults results in variably scar formation. However, there is an extreme variability among individuals in the degree of scarring to similar injury. The mechanisms that underlie this spectrum of scar phenotypes in healthy population are unknown. The objective of our study is to determine morphologic and functional phenotypic differences among healthy patients that produce variable scar phenotypes.

Methods: We collected abdominal skin tissue from female patients undergoing abdominoplasty to remove cesarean scars and compared them with normal skin from the surrounding area. Scars were scored by the Vancouver Scar Scale and classified into groups of low (score 1-3) and high (score from 6-9) scares. Tissue sections were analyzed using immunohistochemistry for cell proliferation marker Ki67 to determine functional differences in the normal skin and scar samples of low and high scar-forming patients.

Results:Our biobank is comprised of tissues from n=7 low scar, n=4 high scar forming patients. Preliminary analysis demonstrated that low-scar producing patients had 3.8 fold more proliferating keratinocytes in their normal skin than high-scar patients (12%+/- 2.6 vs. 3.17%+/-2.1 Ki67 positive cells to total epidermis cells; n=2 patients per group, 3 serial sections were analyzed). Similar differences were observed in the scar tissue of low- vs. high-scar patients’ scars (12.4% vs. 6.2%+/-1.4 Ki67 positive cells to total epidermis cells).

Conclusion:These data suggest that fundamental differences in keratinocyte proliferation among patients may be related to the patient propensity for scar formation. It implies keratinocyte proliferation plays important roles in wound closure (i.e. establishment of a barrier), regulates cytokine and ECM production in wound healing and scar maturation.

 

86.09 The extent of scar formation is characterized by changes in keratinocyte proliferation

Posted on January 31, 2019

H. Li1, S. Keswani1  1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Stanford University,Infectious Diseases And Microbiology And Immunology,Palo Alto, CA, USA 3University Of California – Los Angeles,Pediatrics,Los Angeles, CA, USA

Introduction:
The normal course of wound healing in adults results in variably scar formation. However, there is an extreme variability among individuals in the degree of scarring to similar injury. The mechanisms that underlie this spectrum of scar phenotypes in healthy population are unknown. The objective of our study is to determine morphologic and functional phenotypic differences among healthy patients that produce variable scar phenotypes.

Methods:
We collected abdominal skin tissue from female patients undergoing abdominoplasty to remove cesarean scars and compared them with normal skin from the surrounding area. Scars were scored by the Vancouver Scar Scale and classified into groups of low (score 1-3) and high (score from 6-9) scares. Tissue sections were analyzed using immunohistochemistry for cell proliferation marker Ki67 to determine functional differences in the normal skin and scar samples of low and high scar-forming patients.

Results:
Our biobank is comprised of tissues from n=7 low scar, n=4 high scar forming patients. Preliminary analysis demonstrated that low-scar producing patients had 3.8 fold more proliferating keratinocytes in their normal skin than high-scar patients (12%+/- 2.6 vs. 3.17%+/-2.1 Ki67 positive cells to total epidermis cells; n=2 patients per group, 3 serial sections were analyzed). Similar differences were observed in the scar tissue of low- vs. high-scar patients’ scars (12.4% vs. 6.2%+/-1.4 Ki67 positive cells to total epidermis cells).

Conclusion:
These data suggest that fundamental differences in keratinocyte proliferation among patients may be related to the patient propensity for scar formation. It implies keratinocyte proliferation plays important roles in wound closure (i.e. establishment of a barrier), regulates cytokine and ECM production in wound healing and scar maturation.
 

86.05 Exosomes as Mediators of the Fibrotic Response of Dermal Fibroblasts to Biomechanical Tension

Posted on January 31, 2019

H. V. Vangapandu1, N. Templeman1, D. Colchado1, A. Blum1, H. Li1, X. Wang1, E. Steen1, P. Bollyky2, S. Keswani1, M. Robertson1, C. Coarfa1, S. Balaji1  1Baylor College Of Medicine,Dept Of Surgery,Houston, TX, USA 2Stanford University,Palo Alto, CA, USA

Introduction: Wound responses involve fibroblast(FB)-mediated scar formation potentiated by mechanical tension. There is significant heterogeneity in how different people scar from similar injuries. Exosomes play an important role in cell communication which governs scarring. It is unknown if heterogeneous scarring is attributable to differences between FBs, how they respond to tension, and their intercellular communication. We hypothesize that there are differences in FB responses to tension that contribute to scar heterogeneity via exosomes.

Methods: Skin and paired scar tissue was obtained from women with C-section scars who underwent abdominoplasty. Scars were classified as "low" or "high" based on VSS (<3 vs. >6). Fetal skin was evaluated as non-scarring control. Sections were histologically stained (H&E; Trichrome). FBs were isolated from normal skin and scar, cultured on silicone membranes +/-10% static strain (24h), and evaluated for differences in proliferation(Ki67), fibrogenic potential(aSMA, fibrosis array) and genes implicated in exosome biogenesis(RAB27a-b;SMPD3). Exosomes from different scar phenotypes were analyzed(size-Zetasizer; NGS), and adoptively transferred into SCID murine skin wounds, and wound repair was evaluated. p-values by ANOVA; (n=3/group).

Results: High-scarring patient scars had thicker collagen bundles with interstitial FBs in dermis. These FBs also had a significantly higher proliferation rate(p<0.05) in vitro as compared to low and non-scarring FBs. PCR-array results showed that twice as many fibrotic genes were altered in scarFB vs skinFB in high-scar patients as compared to low-scar patients. Rab27a-b and SMPD3 expression was different in non-, low- vs. high-scarring patients skinFB. In low-scar patients, gene expression was similar between normal skinFB and scarFB, where as in the high-scar patients, Rab27a and SMPD3 levels were lower in the scarFB as compared to skinFB. Size distribution profile of exosomes was similar in skin and scarFB of low-scar patients. High-scar FBs produced exosomes larger in size and greater in abundance (p<0.05). NGS showed that there are baseline differences in small ncRNA of different FB-derived exosomes. While tension induced differential changes in a-SMA, Rab27a-b and SMPD and exosome size and cargo profiles in skinFB of different patients, notably, tension-induced expression of the normal skinFB was similar to its paired scarFB baseline phenotype. Lastly, non-scarring FB-derived exosomes resulted in regenerative wound phenotype in SCID dorsal wounds, whereas low and high-scar FB derived exosomes induced a corollary severity in scarring.

Conclusion: Our findings suggest that fibroblasts from different scarring phenotypes respond differentially to tension, including changes in exosome production that could influence fibrogenic phenotype and maintain the scar memory. Mining these exosome profiles will lead to novel scar therapeutics.

 

86.01 Regulating Hyaluronan Deposition Attenuates Tubulointerstitial Fibrosis in Ureteral Obstruction

Posted on January 31, 2019

X. Wang1, S. Balaji1, H. Li1, E. Steen1, P. Bollyky3, J. Cheng2, S. Keswani1  1Baylor College Of Medicine,Pediatric Surgery/Surgery,Houston, TX, USA 2Baylor College Of Medicine,Nephrology/Medicine,Houston, TX, USA 3Stanford University,Infectious Diseases/Medicine,Palo Alto, CA, USA

Introduction: Renal fibrosis is a pathological characteristic of chronic kidney disease (CKD), and is a product of aberrant inflammation, extracellular matrix (ECM) deposition and peritubular capillary loss. We have demonstrated a novel role for interleukin-10 (IL-10) in abrogating dermal fibrosis by regulating hyaluronan (HA) through dermal fibroblasts. We therefore hypothesized that hyaluronan attenuate renal fibrosis via its molecular weight variation to influence extracellular matrix remodeling, promoting angiogenesis and reducing inflammation. 

Methods: In vivo: We performed unilateral ureteral obstruction(UUO) as a renal fibrosis model with/without IL-10 overexpression through the injection under the kidney capsule with normal diet or diets with 5% HA inhibitor, 4-methylumbelliferone(4MU). UUO/sham kidneys were collected at d3, d7, d21 for RNA, ELISA, and/or immunohistochemistry. HA synthesis and degradation enzyme levels were assessed by qPCR and Western blot. HA molecular weight was assessed by size-exclusion chromatography. In vitro: Renal fibroblasts (FB) were isolated from C57BL/6J mice to determine the effect of IL-10(100 ng/ml) on gene expression of HAS1-3 and hyaluronidases (HYAL1-2) at 24h.  Alpha-SMA, HAS1, HAS2 and p-STAT3 expression were assessed by western blotting at 48h. Data mean+/-SD; p-values by ANOVA. 

Results:
In vivo, the up-regulation of HAS1 and HAS2 expression in normal and 4-MU diets UUO mice compared to control mice from day 3; ELISA showed that total HA is steady increased from day 3 up to day 14 for UUO mice, and up to day 21 for IL-10 treated UUO mice. In normal diet mice, lenti-IL-10 treatment resulted in less dilated tubules, decreased kidney fibrosis, and preserved tubular integrity in kidneys, compared to control treated mice; IL-10 treated 4-MU diet mice were not able to achieve attenuated fibrosis.). HA gel electrophoresis showed Day 3, 7 and 14 UUO kidneys have a previously unreported 1.5×106kD HA variant compared to control/sham kidneys. In vitro: HAS1, 2&3 and HYAL1 in renal FB (6.62±0.89, 1.83±0.54, 1.84±0.92) was also significantly dysregulated (p<0.05) after 24h IL-10 treatment by qPCR.With western blotting, HAS2, a-SMA and STAT3 were significantly upregulated. A 1.88-fold increase in HA-rich matrix formation was shown with 24h IL-10 stimulation, and the effect was abrogated by HYAL (p<0.05). 

Conclusion:

Our study provides the first evidence that injured kidney tissues have the capacity to express increased levels of a high molecular weight HA variant, which contrasts to that found in normal, uninjured kidneys. This finding suggesting that HA plays important roles in kidney, development, homeostasis, architectural integrity and function.Moreover, our discovery of mechanisms behind the HA-attenuated fibrosis could inspire novel therapeutics.

 

82.10 A Fetal 3D Surgical Simulator of Minimally Invasive In Utero Gastroschisis Repair

Posted on January 31, 2019

E. H. Steen1, J. Fisher1,3, O. Olutoye1,3, J. Zaneveld4, N. Salas1, T. Lee1,3, S. Keswani1,3  1Baylor College Of Medicine,Department Of Surgery,Houston, TX, USA 3Texas Children’s Hospital,Division Of Pediatric Surgery,Houston, TX, USA 4Lazarus 3D,Houston, TX, USA

Introduction: We have reported the clinical benefits of fetal minimally invasive surgery (MIS) in attenuating preterm labor, uterine morbidity, and subsequent C-sections – complications associated with open fetal surgery. Other non-lethal diseases may also benefit from fetal MIS, such as gastroschisis. 3D printing allows the creation of lifelike human models. The aim of this study is developing and validating a 3D fetal MIS model to test an in utero procedure for gastroschisis repair.

Methods: A 3D reconstruction of a uterus and fetus with gastroschisis (based on a mid-gestation fetal MRI) was optimized (3D Slicer) and rapidly prototyped using a next-gen Lazarus 3D printer. A four-step MIS procedure (evaluation of fetus, evaluation of bowel, reduction of bowel, coverage of defect) was designed and time-tested in three cohorts repeated in triplicate (fetal/neonatal surgeons, residents, and students, n=6/group). A ten question post-trial validation survey was administered to the participants. Data is presented as mean +/-SD, analysis by ANOVA, post-hoc Tukey HSD, p<0.05.

Results: All procedures were completed successfully (n=54). Operative time was significantly related to surgical training level (fetal/neonatal surgeons 125s+/-29s, residents 141s+/-30s, students 376s+/-107s; p<0.05) with sequential attempts yielding significant rates of improvement in all cohorts. All surgeons reported that the model 1) is an accurate tactile and visually representative model, 2) adequately assessed technical skills required for the procedure, and 3) would be a valuable training tool. The cost for this model was $68.69/trial and can be refurbished/reused for $200.

Conclusion:Our data supports construct, content, and face validity of a novel 3D fetal surgical simulator. This model is more cost effective than animal models in developing fetal techniques and seems to be more representative of the human disease. With the attenuation of maternal-fetal risk observed in fetal MIS, in utero therapies for gastroschisis may be considered.

 

46.15 Tension Influences Small ncRNA Regulatory Landscape of MSC-derived Exosomes During Wound Healing

Posted on January 31, 2019

D. Colchado1, H. V. Vangapandu1, N. Templeman1, H. Li1, Y. Ning1, A. Blum1, P. Bollyky2, S. Keswani1, M. Robertson1, C. Coarfa1, S. Balaji1  1Baylor College Of Medicine,Houston, TX, USA 2Stanford University,Palo Alto, CA, USA

Introduction: Mesenchymal stem cells (MSCs) have a huge therapeutic potential in wound healing. While it is known that the extracellular environment affects the MSC secretome, the role of mechanical tension on the bioactive extracellular vesicles, namely exosomes, released by MSCs, is not known. We hypothesized that mechanical tension regulates MSC exosome production and influences wound healing via paracrine effects on dermal fibroblasts.

Methods: Human MSCs were cultured on silicone membranes +/-10% tonic strain for 24h and analyzed for phenotypic changes (morphology, alpha-SMA, and fibrosis PCR-array) and genes important in exosome biogenesis  (RAb27a-b;SMPD3). Exosomes were isolated and analyzed for size and quantity (Zetasizer). The exosome protein level was quantified (BCA Assay) and Westernblotting (CD63,HSP70,CD9) and Next-Gen Sequencing were performed. Exosomes were labeled by Exo-Glow before use in a primary human dermal fibroblast (FB) migration assay. p-values by ANOVA; (n=3/group).

Results:Tension resulted in the loss of the characteristic morphology of the MSC spindle shape and increased alpha-SMA staining in MSCs. There was a significant change (>2-fold) in ~ 30/77 fibrotic genes with tension. Tension upregulated the expression of IL-10 and IL13RA2, which are also involved in anti-inflammatory cytokine processes. In contrast, pro-fibrotic and pro-inflammatory genes such as Acta-2 and Ccl-2 were downregulated by tension. Additionally, genes encoding fibrinolytic enzymes such as PLAT and growth factors (EGF, VEGFA, and CTGF) were downregulated under tension. Tension downregulated the expression of both RAb27a-b (p<0.01) in MSCs, and there was a corresponding phenotype of perinuclear reorganization of exosomes with an increase in size distribution and protein levels under tension (p<0.05). The three exosome surface markers were verified by Western blotting. Exosomes were enriched for small RNAs as expected. The abundance of tRNA was increased, whereas the miRNAs and lincRNAs in the MSC-derived exosomes were reduced under tension. KEGG analysis of the gene targets and pathways of down regulated miRNAs showed enrichment of intracellular and extracellular wound healing processes. Interestingly, MSC-derived exosomes under static conditions slowed the migration of FB in a scratch wound assay, whereas those derived under tension increased FB migration (p<0.05), but there was no effect of the complete MSC-conditioned media from either static or tension conditions on FB migration.

Conclusion:Mechanical tension induces a fibrogenic and inflammatory phenotype in MSCs. Given that the production and composition of bioactive cargo in MSC exosomes is regulated by tension and can influence FB behavior, we propose that MSC-derived exosomes are a likely target for extracellular communication during wound healing. These insights provide a key role in the development of exosome-based clinical therapies in the context of wound healing and fibrosis.

46.14 A Scar Scoring Method to Predict Recurrence of Discrete Subaortic Stenosis After Surgical Resection

Posted on January 31, 2019

Y. NING1, M. Fahrenholtz1, K. Brown2, E. Steen1, M. Kang2, L. Masri1, L. Wadhwa1, S. Balaji1, K. Grande-Allen2, S. Keswani1  1Texas Children’s Hospital,Pediatrics,Houston, TX, USA 2Rice University,Bioengineering,Houston, TX, USA

Introduction:

Discrete subaortic stenosis (DSS) features the formation of an obstructive fibrous tissue lesion in the left ventricular outflow tract, and while the standard of care is surgical resection, lesions recur in 25-30% of all patients, delineating an “aggressive” phenotype. Here, we define unique histologic parameters that discriminate non-aggressive from aggressive DSS lesions and are consistent with preoperative LVOT gradients, which are known prognostic signs of recurrence.

Methods:

Human DSS patient tissues (n=7) were obtained from the TCH Congenital Heart Biorepository in accordance with IRB protocols. We compared tissue samples from 4 non-aggressive and 3 aggressive recurrent lesions from DSS patients by immunohistochemical staining of cardiac fibroblast DDR2. Next, cell density (H&E), mature/immature collagen ratio (Herovici), and percent DDR2+ cells/HPF were quantified. Lastly, each variable tested was normalized to the ranged set and then combined to generate a DSS aggressiveness score (DAS). Preoperative echocardiographic reports were obtained for all DSS patients whose tissues were analyzed and assessed for mean LVOT gradient. Data was reported as mean ± standard deviation. Statistical analysis was performed using Student’s t-test in which p ≤ 0.05 values were considered significant.

Results:

A differential DDR2 staining was observed between non-aggressive tissues (36.3±27.4% positive/field area) and aggressive tissues, which had almost no measurable expression of DDR2 (1.94±1.90%, p < 0.05). DSS tissues stained by H&E showed significant differences in cell density between aggressive (130±28 cells/image field) and non-aggressive lesions (50±15 cells/field; p < 0.01). Although we observed high variability in both groups, aggressive tissues had increased immature to mature collagen ratios in comparison to non-aggressive ones. Qualitatively, aggressive tissues were more likely to have thin, highly disorganized collagen bundles, whereas non-aggressive tissues had more and thicker collagen bundles. Overall, there was a significant DAS difference between aggressive and non-aggressive tissues (1.94 ±0.46 vs 0.59 ±0.48, p < 0.01) in which the score correlated with the recurrence prediction based on LVOT gradient. Our scoring model will be validated by larger patient sample cohorts.

Conclusion:

Our data underscore histologic features that discriminate aggressive from non-aggressive DSS tissues, which can be quantitatively converted to produce score values that correlate with known predictors of recurrence. This score system may provide a useful means to improve monitoring of aggressive DSS patients.

 

 

44.05 A Thermogelling Hyaluronic Acid Vaginal Stent to Reduce Postoperative Vaginal Fibrosis

Posted on January 31, 2019

J. Hakim1, O. Wyman2, S. Keswani1  1Texas Children’s Hospital,Pediatric Adolescent Gynecology,Houston, TX, USA 2Baylor College Of Medicine,Houston, TX, USA

Introduction:  There is a need for new therapies to prevent vaginal fibrosis. Up to 50,000 girls and 213,000 adult women yearly in the United States require vaginal reconstructive surgery. There is a high rate (up to 73%) of vaginal fibrosis after these surgeries. Sequelae from vaginal fibrosis can be life-long and lead to significant reductions in quality of life. Currently, treatments are limited to conjugated estrogen (CEE) creams in combination with vaginal stents. Unfortunately, current CEE creams cannot assess delivered estrogen due to cream egress from the vagina and the cumbersome nature of vaginal stents leads to early discontinuation. We have created a novel hydrogel that utilizes “click” chemistry to make a thermogelling hyaluronic acid (HA) drug eluting hydrogel. We sought to compare local estrogen delivery with Premarin cream to sustained estrogen delivery with a novel CEE-eluding NorbHA hydrogel on vaginal wound healing.

Methods:  Norbornene-functionalized hyaluronic acid (NorbHA) and tetrazine-functionalized hyaluronic acid (TetHA) was synthesized. Hydrogel was composed of a 1:1 ratio of NorbHA:TetHA with various concentrations of estradiol (E2), 1.5 MDa HA, or a combination of both. The vaginal stents were placed into a full-thickness 1 mm murine vaginal excisional injury model. Animals were sacrificed at days 0, 2, 3, 7, 10. Macrophage inhibiting factor 1 (MIF1), transforming growth factor-β3 (TGFβ3) expression and vascular endothelial growth factor (VEGF) expression were analysed as markers of inflammation and angiogenesis respectively. Histology and immunohistochemistry were used to assess wound resolution and estrogen receptor (ER) density.

Results: Dissolution parameters demonstrated that drug release was still possible until 72h in the thermogelling hydrogel compared to CEE cream (12h). There was a burst release of E2 over the first 12h period followed by a steady-state release while HA had a burst release at 24 h. A statistically significant decrease in MIF1 expression was found in the group containing both HA and E2 compared to the CEE cream between the day 0 and day 3 timepoints. Greater expression of TGFβ3 and VEGF were found in the groups containing E2, or E2/HA compared to the estrogen cream by day 2. ER density increases with E2 delivery but appears to be dependent on circulating estrogen levels. 

Conclusion: Vaginal tissue healing is enhanced through a novel thermogelling vaginal hydrogel with sustained estrogen and HA release compared to local exogenous estrogen cream. Improvements in both the anti-inflammatory and pro-angiogenic effects of sustained estrogen delivery included reduction in neutrophil and macrophage infiltration, modulating ECM degradation and stabilizing collagen. Further development of this platform may provide a substantial increase in efficacy of E2 delivery to the vaginal tissue and reduce post-operative vaginal fibrosis.

 

18.20 Central venous catheter-related DVT in the pediatric CVICU: causes and complications

Posted on January 31, 2019

E. H. Steen1, J. J. Lasa3, T. C. Nguyen3, S. G. Keswani2, P. A. Checchia3, M. M. Anders3  1Baylor College Of Medicine,Department Of Surgery,Houston, TX, USA 2Texas Children’s Hospital,Division Of Pediatric Surgery, Department Of Surgery,Houston, TX, USA 3Texas Children’s Hospital,Section Of Critical Care Medicine, Department Of Pediatrics,Houston, TX, USA

Introduction: Central venous catheter (CVC) use is common in the management of critically ill children, especially those with congenital or acquired heart disease (CHD). Prior reports suggest that the presence of a CVC augments the risk of deep vein thrombosis (DVT). How CVC-associated DVTs contribute to morbidity and mortality in this high risk patient population is unknown. Taken together, the aim of this study is to identify the factors associated with DVT and thrombus propagation in the pediatric cardiovascular intensive care unit (CVICU) population.

Methods: The PC4 database and a radiologic imaging database for patients admitted to Texas Children’s Hospital were retrospectively reviewed. During the one year study period (January – December 2017), there were 1215 unique central lines placed in 851 admissions. Information gathered included demographics and outcomes of patients requiring central line placement in the TCH CVICU, as well as the incidence of DVT and complications. Data shown as OR [95% CI] by univariate linear regression; p value < 0.05 considered significant.

Results: DVT was diagnosed in 8% of admissions with a CVC. Almost 30% of these patients demonstrated thrombus extension into the inferior vena cava (IVC). The diagnosis of DVT is a highly significant risk factor for mortality in these patients (p=.0001, OR 6.1 [2.8, 13.1]). In a univariate regression model, the risk factors most significantly associated with DVT include the presence of more than one line and higher total line hours (defined as the sum of all lines multiplied by the number of hours each line was in place), as well as longer duration of intubation and extended CVICU admission times. A diagnosis of low cardiac output syndrome (LCOS), sepsis, UTI, CLABSI, and cardiac catheterization during admission are also significant risk factors. Of these, only longer catheter dwell times (p=.0001) and cardiac catheterization (p=.002) are significantly associated with the diagnosis of DVT on multivariate analysis. Interestingly, both LCOS and CLABSI (p<0.0001 in each) are significantly associated with propagation of the thrombus into the IVC. Of note, cardiac surgery with cardiopulmonary bypass appears to be protective of clot development (p=0.001, OR 0.38 [0.22, 0.67]). 

Conclusion: We have defined risk factors for CVC-associated DVT in the pediatric CVICU population, as well as specific factors associated with clot propagation into the IVC. CVC-associated DVTs impart a significant risk of morbidity and mortality in critically ill children, highlighting the need for well-designed studies to determine the best preventive and therapeutic strategies and to establish guidelines for appropriate monitoring and follow up of these patients.

14.17 Not All Bilateral Congenital Diaphragmatic Hernias are the Same

Posted on January 31, 2019

H. Sriraman1, M. Verla1, C. Style1, A. Mehollin-Ray2, C. Fernandes3, A. Vogel1, T. Lee1, S. Keswani1, O. Oluyinka1  1Baylor College Of Medicine, Michael E. DeBakey Department of Surgery,Texas Children’s Fetal Center,Houston, TX, USA 2Texas Children’s Hospital – Fetal Center,Department Of Radiology,Houston, TX, USA 3Texas Children’s Hospital,Departments Of Pediatrics – Newborn Section,Houston, TX, USA

Introduction:  Bilateral congenital diaphragmatic hernia (CDH) is a rare variant of CDH with a mortality rate as high as 74%. However, we hypothesize that not all variants of bilateral CDH have a poor prognosis. The aim of our study was to evaluate our institution’s postnatal outcomes of neonates with bilateral congenital diaphragmatic hernia to determine which elements may portend a better prognosis.

Methods:  Following IRB approval, a single center, retrospective review of all patients with bilateral CDH evaluated from January 2004 to December 2017 was performed. Demographics, associated congenital abnormalities, type of CDH defect, operative repair and approach, ECMO use and survival were collected. Descriptive statistics were used to analyze the data.

Results: Over the 14-year study period, 282 patients with CDH were identified and 7 had a bilateral intrapleural defect. Six of the seven neonates with bilateral CDH were diagnosed prenatally. Four neonates had posterior-lateral defects, while the other three neonates had anterior CDH defects. Three neonates had at least one concomitant major congenital anomaly, but none had a genetic anomaly (Table 1). The median gestational age at birth was 38 weeks (IQR: 37, 39) and birthweight was 3020 grams (IQR: 2288, 3525). The median Apgar scores at 1 and 5 minutes were 6 (IQR: 3, 7) and 8 (IQR: 7, 8), respectively. None of the seven patients required ECMO and the overall cohort survival was 57% (Table 1). Median age at follow-up was 2 years (IQR: 0.4, 7). All the patients with anterior defects received a primary repair, while two of the four neonates with posterior-lateral defects had some form of patch repair.

Conclusion: Prognosis from bilateral intrapleural CDH may not be as grim as initially reported. Compared to posterior-lateral defects, patients with large bilateral anterior defects have a relatively benign course. This case series indicates good outcomes for patients with bilateral anterior CDH defects which suggests that not all patients with bilateral CDH will have a dismal outcome. Better prenatal determination of anterior versus posterior bilateral CDH may be helpful when counseling about clinical outcomes.

14.13 Predictors and Outcomes of Tracheostomy Patients with Congenital Diaphragmatic Hernia

Posted on January 31, 2019

C. C. Style1, M. A. Verla1, T. C. Lee1, S. C. Fallon1, H. Srirani1, C. J. Fernandes2, S. G. Keswani1, A. M. Vogel1, O. O. Olutoye1  1Baylor College Of Medicine, Texas Childrens Hospital,Department Of Surgery, Divison Of Pediatric Surgery,Houston, TX, USA 2Baylor College Of Medicine, Texas Children’s Hospital,Department Of Pediatrics, Newborn Secton,Houston, TX, USA

Introduction: Numerous advances in pre- and postnatal management of critically-ill CDH patients have improved long term survival and outcomes, but a small percentage of patients continue to require tracheostomy.  The purpose of this study was to define the characteristics and associated risk factors for tracheostomy in the CDH population. 

Methods: An IRB approved retrospective review of all infants evaluated for CDH at a single institution from March 2004 to April 2018 was performed. Data analyzed included maternal and fetal demographics, prenatal imaging data, and postnatal clinical outcomes.  Primary outcomes assessed were indication for and duration of tracheostomy, requirement for ECMO and survival.  Statistical analysis included chi-square analysis, student’s t test, and stepwise logistic regression.  A p-value of <0.05 was considered significant. 

Results:Of 273 CDH patients treated, 10% (26) underwent a tracheostomy prior to 2 years of life (median age of 4 [3 – 8] months). Of these 26 patients, 65% (17) had a left-sided CDH and 76% (19) were male.  Indications for tracheostomy were persistent pulmonary hypertension in the setting of severe pulmonary hypoplasia (31%, 8), tracheomalacia (27%, 7), bronchopulmonary dysplasia (27%, 7), upper airway obstruction/structural defect (11%, 3), and vocal cord paralysis (4%, n=1).  Only two patients underwent tracheostomy after their initial inpatient hospitalization; both were eventual recipients of lung transplants.  Prenatally, although lungs volumes were similar to the non-tracheostomy cohort, percent liver herniation was significantly higher in the tracheostomy group (27% ± 18, p<0.01).  Additionally, 79%(p=0.009) had an associated structural, genetic, and/or cardiac anomaly, which was a strong predictor of tracheostomy (OR 4.991,CI:1.25 – 20.3) in this cohort.  At birth, prematurity and low birthweight also significantly correlated to need for tracheostomy (p<0.05, table 1). Incidence of ECMO was similar to the non-tracheostomy cohort (39% vs 31%, p=0.44) as was the overall survival (62% vs 74%, p=0.171). Non-survivors with a tracheostomy were, however, significantly older at time of death. Of the surviving tracheostomy patients (n=16), median length of hospital stay was prolonged 259 [187, 299] days (p<0.05) with a median time from tracheostomy to discharge of 98 [78, 172] days.  Length of outpatient follow-up was 3 [0.5 – 9] years during which time 38% of survivors were decannulated (a range of 28 days to 3.5 years after tracheostomy).

Conclusion:CDH patients born prematurely and/or those with associated anomalies are at increased risk for tracheostomy.  This is associated with prolonged hospitalization and median tracheostomy time of 1.5 years.  

 

13.12 Cholestatic Hyperbilirubinemia in Infants with Congenital Diaphragmatic Hernia

Posted on January 31, 2019

C. A. Ikedionwu1, C. C. Style1, M. A. Verla1, T. C. Lee1, C. J. Fernandes2, S. G. Keswani1, A. M. Vogel1, O. O. Olutoye1  1Baylor College Of Medicine, Texas Childrens Hospital,Department Of Surgery, Division Of Pediatric Surgery,Houston, TX, USA 2Baylor College Of Medicine, Texas Childrens Hospital,Department Of Pediatrics, Newborn Section,Houston, TX, USA

Introduction:  Hyperbilirubinemia is often present in neonates with congenital anomalies, particularly congenital diaphragmatic hernia (CDH). Hence, we sought to identify risk factors for hyperbilirubinemia and the relationship with outcome in children with CDH.

Methods: An IRB approved retrospective review of all infants admitted with CDH at a single institution from January 2012 to December 2017 was performed. The cohort was categorized by the presence or absence of hyperbilirubinemia defined as either a total serum bilirubin (TSB) of >5mg/dL, direct bilirubin (DB) >1.5mg/dL or DB >20% of TSB. Patients were further stratified into physiologic vs cholestatic (conjugated) hyperbilirubinemia based on normograms for bilirubin levels and neonatal age. Data analyzed included demographics, prenatal imaging features, and postnatal clinical outcomes. Data were analyzed using chi-square, Mann-Whitney U test and Student’s t-test, and logistic regression as appropriate; a p-value of <0.05 was considered significant.

Results: A total of 122 CDH patients were identified, of which 75% were left-sided, 53% were male, and hyperbilirubinemia (HB) developed in 69% (84). The primary HB subtype, physiologic hyperbilirubinemia (PHB) was noted in 35% (43/122), while cholestatic HB (CHB) accounted for the remaining 34% (41/122). The majority of those with HB development had “liver up” morphology (81% (68/84), p=0.012) and all received total parenteral nutrition (TPN). Compared to infants with PHB, infants with CHB had prenatal findings with smaller lung volumes (O/E TFLV of 28% vs 38% p=0.007), albeit without significant difference in percent liver herniation. Postnatally, those with CHB were more likely to have had a patch repair, longer duration of TPN, developed sepsis and needed ECMO when compared to both infants with PHB and those without evidence of HB (p<0.05, Table 1). Of these variables, logistic regression revealed that ECMO was the foremost risk factor for the development of CHB (OR 0.161; CI:0.31-0.84].  Infants with CHB had a longer length of hospital stay and lower survival compared to those with PHB. However, there was no difference in the survival between those with CHB and those with no jaundice. In spite of the high incidence of CHB in the cohort, only one patient had findings of hepatic encephalopathy.

Conclusion: CDH infants are high risk for developing clinically significant hyperbilirubinemia. Cholestatic hyperbilirubinemia is associated with increased severity of CDH and portends a worse prognosis. ECMO appears to be a major factor associated with parenteral nutrition associated liver disease (PNALD) in CDH.  Patients with CDH whgo undergo ECMO should be considered for management strategies to reduce PNALD.