Clinical Utility of Laser Speckle Contrast Imaging and Real-Time Quantification of Bowel Perfusion in Minimally Invasive Left-Sided Colorectal Resections.

BACKGROUND: Left-sided colorectal surgery demonstrates high anastomotic leak rates, with tissue ischemia thought to influence outcomes. Indocyanine green is commonly used for perfusion assessment, but evidence remains mixed for whether it reduces colorectal anastomotic leaks. Laser speckle contrast imaging provides dye-free perfusion assessment in real-time through perfusion heat maps and quantification. OBJECTIVE: This study investigates the efficacy of advanced visualization (indocyanine green versus laser speckle contrast imaging), perfusion assessment, and utility of laser speckle perfusion quantification in determining ischemic margins. DESIGN: Prospective intervention group using advanced visualization with case-matched, retrospective control group. SETTINGS: Single academic medical center. PATIENTS: Forty adult patients undergoing elective, minimally invasive, left-sided colorectal surgery. INTERVENTIONS: Intraoperative perfusion assessment using white light imaging and advanced visualization at 3 time points: T1-proximal colon after devascularization, before transection, T2-proximal/distal colon before anastomosis, and T3-completed anastomosis. MAIN OUTCOME MEASURES: Intraoperative indication of ischemic line of demarcation before resection under each visualization method, surgical decision change using advanced visualization, post hoc laser speckle perfusion quantification of colorectal tissue, and 30-day postoperative outcomes. RESULTS: Advanced visualization changed surgical decision-making in 17.5% of cases. For cases in which surgeons changed a decision, the average discordance between the line of demarcation in white light imaging and advanced visualization was 3.7 cm, compared to 0.41 cm ( p = 0.01) for cases without decision changes. There was no statistical difference between the line of ischemic demarcation using laser speckle versus indocyanine green ( p = 0.16). Laser speckle quantified lower perfusion values for tissues beyond the line of ischemic demarcation while suggesting an additional 1 cm of perfused tissue beyond this line. One (2.5%) anastomotic leak occurred in the intervention group. LIMITATIONS: This study was not powered to detect differences in anastomotic leak rates. CONCLUSIONS: Advanced visualization using laser speckle and indocyanine green provides valuable perfusion information that impacts surgical decision-making in minimally invasive left-sided colorectal surgeries. See Video Abstract . UTILIDAD CLNICA DE LAS IMGENES DE CONTRASTE MOTEADO CON LSER Y LA CUANTIFICACIN EN TIEMPO REAL DE LA PERFUSIN INTESTINAL EN RESECCIONES COLORRECTALES DEL LADO IZQUIERDO MNIMAMENTE INVASIVAS: ANTECEDENTES:La cirugía colorrectal del lado izquierdo demuestra altas tasas de fuga anastomótica, y se cree que la isquemia tisular influye en los resultados. El verde de indocianina se utiliza habitualmente para evaluar la perfusión, pero la evidencia sobre si reduce las fugas anastomóticas colorrectales sigue siendo contradictoria. Las imágenes de contraste moteado con láser proporcionan una evaluación de la perfusión sin colorantes en tiempo real a través de mapas de calor de perfusión y cuantificación.OBJETIVO:Este estudio investiga la eficacia de la evaluación de la perfusión mediante visualización avanzada (verde de indocianina versus imágenes de contraste moteado con láser) y la utilidad de la cuantificación de la perfusión con moteado láser para determinar los márgenes isquémicos.DISEÑO:Grupo de intervención prospectivo que utiliza visualización avanzada con un grupo de control retrospectivo de casos emparejados.LUGARES:Centro médico académico único.PACIENTES:Cuarenta pacientes adultos sometidos a cirugía colorrectal electiva, mínimamente invasiva, del lado izquierdo.INTERVENCIONES:Evaluación de la perfusión intraoperatoria mediante imágenes con luz blanca y visualización avanzada en tres puntos temporales: T1-colon proximal después de la devascularización, antes de la transección; T2-colon proximal/distal antes de la anastomosis; y T3-anastomosis completa.PRINCIPALES MEDIDAS DE VALORACIÓN:Indicación intraoperatoria de la línea de demarcación isquémica antes de la resección bajo cada método de visualización, cambio de decisión quirúrgica mediante visualización avanzada, cuantificación post-hoc de la perfusión con láser moteado del tejido colorrectal y resultados posoperatorios a los 30 días.RESULTADOS:La visualización avanzada cambió la toma de decisiones quirúrgicas en el 17,5% de los casos. Para los casos en los que los cirujanos cambiaron una decisión, la discordancia promedio entre la línea de demarcación en las imágenes con luz blanca y la visualización avanzada fue de 3,7 cm, en comparación con 0,41 cm (p = 0,01) para los casos sin cambios de decisión. No hubo diferencias estadísticas entre la línea de demarcación isquémica utilizando láser moteado versus verde de indocianina (p = 0,16). El moteado con láser cuantificó valores de perfusión más bajos para los tejidos más allá de la línea de demarcación isquémica y al mismo tiempo sugirió 1 cm adicional de tejido perfundido más allá de esta línea. Se produjo una fuga anastomótica (2,5%) en el grupo de intervención.LIMITACIONES:Este estudio no tuvo el poder estadístico suficiente para detectar diferencias en las tasas de fuga anastomótica.CONCLUSIONES:La visualización avanzada utilizando moteado láser y verde de indocianina proporciona información valiosa sobre la perfusión que impacta la toma de decisiones quirúrgicas en cirugías colorrectales mínimamente invasivas del lado izquierdo. (Traducción-Dr. Ingrid Melo).

Laser speckle contrast imaging compared with indocyanine green in renal perfusion of a porcine model.

Background: When viewed under near-infrared light, indocyanine green (ICG) signal for kidney perfusion can be utilized in partial nephrectomy. Laser speckle contrast imaging (LSCI) uses coherent light to detect perfusion during real-time laparoscopic surgery. Materials and methods: Laser speckle contrast imaging or ActivSight, an imaging sensor adapter, was used during laparoscopy of an anesthetized porcine kidney model. ActivSight's "perfusion mode" and "quantification mode" displayed the blood flow as a heatmap and numerical signal intensity, respectively. Results: After the upper segmental renal artery was clamped, ICG was seen in the lower pole, and LSCI showed low unit (dark color) quantification and perfusion in the upper pole. Indocyanine green was retained in the lower pole after the upper segmental artery was unclamped, and LSCI perfusion was demonstrated in the entire kidney. Conclusions: Laser speckle contrast imaging is a dye-free, repeatable, real-time adjunct for renal parenchymal perfusion assessment applicable to minimally invasive renal surgery to complement the technology of ICG near-infrared fluorescence and advance digital surgery.

Real-time quantification of bowel perfusion using Laparoscopic Laser Speckle Contrast Imaging (LSCI) in a porcine model.

BACKGROUND/PURPOSE: Real-time quantification of tissue perfusion can improve intraoperative surgical decision making. Here we demonstrate the utility of Laser Speckle Contrast Imaging as an intra-operative tool that quantifies real-time regional differences in intestinal perfusion and distinguishes ischemic changes resulting from arterial/venous obstruction. METHODS: Porcine models (n = 3) consisted of selectively devascularized small bowel loops that were used to measure the perfusion responses under conditions of control/no vascular occlusion, arterial inflow occlusion, and venous outflow occlusion using laser speckle imaging and indocyanine green fluoroscopy. Laser Speckle was also used to assess perfusion differences between small bowel antimesenteric-antimesenteric and mesenteric-mesenteric anastomoses. Perfusion quantification was measured in relative perfusion units calculated from the laser speckle perfusion heatmap. RESULTS: Laser Speckle distinguished between visually identified perfused, watershed, and ischemic intestinal segments with both color heatmap and quantification (p < .00001). It detected a continuous gradient of relative intestinal perfusion as a function of distance from the stapled ischemic bowel edge. Strong positive linear correlation between relative perfusion units and changes in mean arterial pressure resulting from both arterial (R2 = .96/.79) and venous pressure changes (R2 = .86/.96) was observed. Furthermore, Laser Speckle showed that the antimesenteric anastomosis had a higher perfusion than mesenteric anastomosis (p < 0.01). CONCLUSIONS: Laser Speckle Contrast Imaging provides objective, quantifiable tissue perfusion information in both color heatmap and relative numerical units. Laser Speckle can detect spatial/temporal differences in perfusion between antimesenteric and mesenteric borders of a bowel segment and precisely detect perfusion changes induced by progressive arterial/venous occlusions in real-time.

Real-time quantification of intestinal perfusion and arterial versus venous occlusion using laser speckle contrast imaging in porcine model.

PURPOSE: Real-time intraoperative perfusion assessment may reduce anastomotic leaks. Laser speckle contrast imaging (LSCI) provides dye-free visualization of perfusion by capturing coherent laser light scatter from red blood cells and displays perfusion as a colormap. Herein, we report a novel method to precisely quantify intestinal perfusion using LSCI. METHODS: ActivSight™ is an FDA-cleared multi-modal visualization system that can detect and display perfusion via both indocyanine green imaging (ICG) and LSCI in minimally invasive surgery. An experimental prototype LSCI perfusion quantification algorithm was evaluated in porcine models. Porcine small bowel was selectively devascularized to create regions of perfused/watershed/ischemic bowel, and progressive aortic inflow/portal vein outflow clamping was performed to study arterial vs. venous ischemia. Continuous arterial pressure was monitored via femoral line. RESULTS: LSCI perfusion colormaps and quantification distinguished between perfused, watershed, and ischemic bowel in all vascular control settings: no vascular occlusion (p < 0.001), aortic occlusion (p < 0.001), and portal venous occlusion (p < 0.001). LSCI quantification demonstrated similar levels of ischemia induced both by states of arterial inflow and venous outflow occlusion. LSCI-quantified perfusion values correlated positively with higher mean arterial pressure and with increasing distance from ischemic bowel. CONCLUSION: LSCI relative perfusion quantification may provide more objective real-time assessment of intestinal perfusion compared to conventional naked eye assessment by quantifying currently subjective gradients of bowel ischemia and identifying both arterial/venous etiologies of ischemia.

Feasibility and comparison of laparoscopic laser speckle contrast imaging to near-infrared display of indocyanine green in intraoperative tissue blood flow/tissue perfusion in preclinical porcine models.

OBJECTIVE: To determine if laser speckle contrast imaging (LSCI) mitigates variations and subjectivity in the use and interpretation of indocyanine green (ICG) fluorescence in the current visualization paradigm of real-time intraoperative tissue blood flow/perfusion in clinically relevant scenarios. METHODS: De novo laparoscopic imaging form-factor detecting real-time blood flow using LSCI and blood volume by near-infrared fluorescence (NIRF) of ICG was compared to ICG NIRF alone, for dye-less real-time visualization of tissue blood flow/perfusion. Experienced surgeons examined LSCI and ICG in segmentally devascularized intestine, partial gastrectomy, and the renal hilum across six porcine models. Precision and accuracy of identifying demarcating lines of ischemia/perfusion in tissues were determined in blinded subjects with varying levels of surgical experience. RESULTS: Unlike ICG, LSCI perfusion detection was real time (latency < 150 ms: p < 0.01), repeatable and on-demand without fluorophore injection. Operating surgeons (n = 6) precisely and accurately identified concordant demarcating lines in white light, LSCI, and ICG modes immediately. Blinded subjects (n = 21) demonstrated similar spatial-temporal precision and accuracy with all three modes ≤ 2 min after ICG injection, and discordance in ICG mode at ≥ 5 min in devascularized small intestine (p < 0.0001) and in partial gastrectomy (p < 0.0001). CONCLUSIONS: Combining LSCI for near real-time blood flow detection with ICG fluorescence for blood volume detection significantly improves precision and accuracy of perfusion detection in tissue locations over time, in real time, and repeatably on-demand than ICG alone.

Utility and usability of laser speckle contrast imaging (LSCI) for displaying real-time tissue perfusion/blood flow in robot-assisted surgery (RAS): comparison to indocyanine green (ICG) and use in laparoscopic surgery.

BACKGROUND: Utility and usability of laser speckle contrast imaging (LSCI) in detecting real-time tissue perfusion in robot-assisted surgery (RAS) and laparoscopic surgery are not known. LSCI displays a color heatmap of real-time tissue blood flow by capturing the interference of coherent laser light on red blood cells. LSCI has advantages in perfusion visualization over indocyanine green imaging (ICG) including repeat use on demand, no need for dye, and no latency between injection and display. Herein, we report the first-in-human clinical comparison of a novel device combining proprietary LSCI processing and ICG for real-time perfusion assessment during RAS and laparoscopic surgeries. METHODS: ActivSight™ imaging module is integrated between a standard laparoscopic camera and scope, capable of detecting tissue blood flow via LSCI and ICG in laparoscopic surgery. From November 2020 to July 2021, we studied its use during elective robotic-assisted and laparoscopic cholecystectomies, colorectal, and bariatric surgeries (NCT# 04633512). For RAS, an ancillary laparoscope with ActivSight imaging module was used for LSCI/ICG visualization. We determined safety, usability, and utility of LSCI in RAS vs. laparoscopic surgery using end-user/surgeon human factor testing (Likert scale 1-5) and compared results with two-tailed t tests. RESULTS: 67 patients were included in the study-40 (60%) RAS vs. 27 (40%) laparoscopic surgeries. Patient demographics were similar in both groups. No adverse events to patients and surgeons were observed in both laparoscopic and RAS groups. Use of an ancillary laparoscopic system for LSCI/ICG visualization had minimal impact on usability in RAS as evidenced by surgeon ratings of device usability (set-up 4.2/5 and form-factor 3.8/5). LSCI ability to detect perfusion (97.5% in RAS vs 100% in laparoscopic cases) was comparable in both RAS and laparoscopic cases. CONCLUSIONS: LSCI demonstrates comparable utility and usability in detecting real-time tissue perfusion/blood flow in RAS and laparoscopic surgery.

An In Vitro Model of the Efficacy of Breast Implant Irrigant Solutions Against Gram-Negative Infections.

BACKGROUND: In implant-based breast surgery, infections remain a clinically challenging complication. Surgeons often prophylactically address this risk by irrigating the implant at the time of placement. However, there remain few data on the ideal irrigant for gram-negative species. METHODS: The authors assessed the relative efficacy of 10% povidone-iodine, triple-antibiotic solution, Prontosan, Clorpactin, and normal saline (negative control) against 3 gram-negative bacterial backgrounds: Escherichia coli , Pseudomonas aeruginosa , and Proteus species. A laboratory-adapted strain and a clinical isolate were selected for each group of bacteria. Sterile, smooth implant discs were immersed in each irrigant solution and then incubated in suspensions of each bacterial strain overnight at 37°C. Each disc was then rinsed and sonicated to displace biofilm-forming bacteria from the implant surface. The displaced bacteria were enumerated by plating, and normalized values were calculated for the bacterial counts of each irrigant. RESULTS: Povidone-iodine resulted in the greatest reduction of bacterial load for all 6 strains by a factor of 10 1 to 10 6 . Prontosan had a lesser, yet significant reduction in all bacterial strains. Triple-antibiotic solution demonstrated the greatest reduction in one Proteus species strain, and Clorpactin reduced bacterial counts in only half of the bacterial strains. When comparing laboratory strains to clinical isolates, significant differences were seen in each bacterial species in at least 2 irrigant solutions. CONCLUSIONS: Povidone-iodine has been proven the most effective at reducing bacterial contamination of E. coli, P. aeruginosa , and Proteus species in both laboratory-adapted strains and clinical isolates. CLINICAL RELEVANCE: This study proves that povidone-iodine is the most effective at preventing gram-negative infections in breast implant surgery.

Select Whole-Cell Biofilm-Based Immunogens Protect against a Virulent Staphylococcus Isolate in a Stringent Implant Model of Infection.

Many microbes of concern to human health remain without vaccines. We have developed a whole-microbe inactivation technology that enables us to rapidly inactivate large quantities of a pathogen while retaining epitopes that were destroyed by previous inactivation methods. The method that we call UVC-MDP inactivation can be used to make whole-cell vaccines with increased potency. We and others are exploring the possibility of using improved irradiation-inactivation technologies to develop whole-cell vaccines for numerous antibiotic-resistant microbes. Here, we apply UVC-MDP to produce candidate MRSA vaccines which we test in a stringent tibia implant model of infection challenged with a virulent MSRA strain. We report high levels of clearance in the model and observe a pattern of protection that correlates with the immunogen protein profile used for vaccination.

Characterizing placental stiffness using ultrasound shear-wave elastography in healthy and preeclamptic pregnancies.

PURPOSE: To measure the stiffness of the placenta in healthy and preeclamptic patients in the second and third trimesters of pregnancy using ultrasound shear-wave elastography (SWE). We also aimed to evaluate the effect of age, gestational age, gravidity, parity and body mass index (BMI) on placental stiffness and a possible correlation of stiffness with perinatal outcomes. METHODS: In a case-control study, we recruited a total of 47 singleton pregnancies in the second and third trimesters of which 24 were healthy and 23 were diagnosed with preeclampsia. In vivo placental stiffness was measured once at the time of recruitment for each patient. Pregnancies with posterior placentas, multiple gestation, gestational hypertension, chronic hypertension, diabetes, autoimmune disease, fetal growth restriction and congenital anomalies were excluded. RESULTS: The mean placental stiffness was significantly higher in preeclamptic pregnancies compared to controls in the third trimester (difference of means = 16.8; 95% CI (9.0, 24.5); P < 0.001). There were no significant differences in placental stiffness between the two groups in the second trimester or between the severe preeclampsia and preeclampsia without severe features groups (difference of means = 9.86; 95% CI (-5.95, 25.7); P ≥ 0.05). Peripheral regions of the placenta were significantly stiffer than central regions in the preeclamptic group (difference of means = 10.67; 95% CI (0.07, 21.27); P < 0.05), which was not observed in the control group (difference of means = 0.55; 95% CI (- 5.25, 6.35); P > 0.05). We did not identify a correlation of placental stiffness with gestational age, maternal age, gravidity or parity. However, there was a statistically significant correlation with BMI (P < 0.05). In addition, pregnancies with higher placental stiffness during the 2nd and 3rd trimesters had significantly reduced birth weight (2890 ± 176 vs. 2420 ± 219 g) and earlier GA (37.8 ± 0.84 vs. 34.3 ± 0.98 weeks) at delivery (P < 0.05). CONCLUSION: Compared to healthy pregnancies, placentas of preeclamptic pregnancies are stiffer and more heterogeneous. Placental stiffness is not affected by gestational age or the severity of preeclampsia but there is a correlation with higher BMI and poor perinatal outcomes.

Are Current Technical Exclusion Criteria for Clinical Trials of Magnetic Resonance-Guided High-Intensity Focused Ultrasound Too Restrictive?: Early Experiences at a Pediatric Hospital.

Certain technical criteria must be met to ensure the treatment safety of magnetic resonance-guided high-intensity focused ultrasound. We retrospectively reviewed how our enrollment criteria were applied from 2014 to 2017 in a clinical trial of magnetic resonance-guided high-intensity focused ultrasound ablation of recurrent malignant and locally aggressive benign solid tumors. Among the 36 screened patients between 2014 and 2017, more than one-third were excluded for technical exclusion criteria such as the anatomic location and proximity to prosthetics. Overall, patients were difficult to accrue for this trial, given the incidence of these tumors. To increase potential accrual, screening exclusion criteria could be more generalized and centered on the ability to achieve an acceptable treatment safety margin, rather than specifically excluding on the basis of general anatomic areas.

High-Intensity Focused Ultrasound (HIFU) Triggers Immune Sensitization of Refractory Murine Neuroblastoma to Checkpoint Inhibitor Therapy.

PURPOSE: Immunotherapy promises unprecedented benefits to patients with cancer. However, the majority of cancer types, including high-risk neuroblastoma, remain immunologically unresponsive. High-intensity focused ultrasound (HIFU) is a noninvasive technique that can mechanically fractionate tumors, transforming immunologically "cold" tumors into responsive "hot" tumors. EXPERIMENTAL DESIGN: We treated <2% of tumor volume in previously unresponsive, large, refractory murine neuroblastoma tumors with mechanical HIFU and assessed systemic immune response using flow cytometry, ELISA, and gene sequencing. In addition, we combined this treatment with αCTLA-4 and αPD-L1 to study its effect on the immune response and long-term survival. RESULTS: Combining HIFU with αCTLA-4 and αPD-L1 significantly enhances antitumor response, improving survival from 0% to 62.5%. HIFU alone causes upregulation of splenic and lymph node NK cells and circulating IL2, IFNγ, and DAMPs, whereas immune regulators like CD4+Foxp3+, IL10, and VEGF-A are significantly reduced. HIFU combined with checkpoint inhibitors induced significant increases in intratumoral CD4+, CD8α+, and CD8α+CD11c+ cells, CD11c+ in regional lymph nodes, and decrease in circulating IL10 compared with untreated group. We also report significant abscopal effect following unilateral treatment of mice with large, established bilateral tumors using HIFU and checkpoint inhibitors compared with tumors treated with HIFU or checkpoint inhibitors alone (61.1% survival, P < 0.0001). This combination treatment significantly also induces CD4+CD44+hiCD62L+low and CD8α+CD44+hiCD62L+low population and is adoptively transferable, imparting immunity, slowing subsequent de novo tumor engraftment. CONCLUSIONS: Mechanical fractionation of tumors using HIFU can effectively induce immune sensitization in a previously unresponsive murine neuroblastoma model and promises a novel yet efficacious immunoadjuvant modality to overcome therapeutic resistance.

Assessing SSRIs' effects on fetal cardiomyocytes utilizing placenta-fetus model.

Selective serotonin reuptake inhibitors (SSRIs) have been shown to hinder cardiomyocyte signaling, raising concerns about their safety during pregnancy. Approaches to assess SSRI-induced effects on fetal cardiovascular cells following passage of drugs through the placental barrier in vitro have only recently become available. Herein, we report that the SSRIs, fluoxetine and sertraline, lead to slowed cardiomyocyte calcium oscillations and induce increased secretion of troponin T and creatine kinase-MB with reduced secretion of NT-proBNP, three key cardiac injury biomarkers. We show the cardiomyocyte calcium handling effects are further amplified following indirect exposure through a placental barrier model. These studies are the first to investigate the effects of placental barrier co-culture with cardiomyocytes in vitro and to show cardiotoxicity of SSRIs following passage through the placental barrier. STATEMENT OF SIGNIFICANCE: Use of selective serotonin reuptake inhibitors (SSRIs), a class of antidepressants, during pregnancy continues to rise despite multiple studies showing potential for detrimental effects on the developing fetus. SSRIs are particularly thought to slow cardiovascular electrical activity, such as ion signaling, yet few, if any, methods exist to rigorously study these drug-induced effects on human pregnancy and the developing fetus. Within this study, we utilized a placenta-fetus model to evaluate these drug-induced effects on cardiomyocytes, looking the drugs' effects on calcium handling and secretion of multiple cardiac injury biomarkers. Together, with existing literature, this study provides a platform for assessing pharmacologic effects of drugs on cells mimicking the fetus and the role the placenta plays in this process.

Model Placental Barrier Phenotypic Response to Fluoxetine and Sertraline: A Comparative Study.

Medications taken during pregnancy may significantly impact fetal development, yet there are few studies that rigorously assess medication safety due to ethical concerns. Selective serotonin reuptake inhibitors (SSRIs) are a class of drug increasingly being prescribed for depression, yet multiple studies have shown that taking SSRIs during pregnancy can lead to preterm birth and potential health concerns for the baby. Therefore, a biomimetic placental barrier model is utilized herein to assess transport profiles and phenotypic effects resulting from SSRI exposure, comparing fluoxetine and sertraline. Results show that the placental barrier quickly uptakes drug from the maternal side, but slowly releases on the fetal side. Phenotypically, there is a dose-dependent change in cell adhesion molecule (CAM) and transforming growth factor beta (TGFß) secretions, markers of cell adhesion and angiogenesis. Both drugs impact CAM secretions, whereas sertraline alone impacts TGFß secretions. When evaluating cell type, it becomes clear that endothelial cells, not trophoblast, are the main cell type involved in these phenotypic changes. Overall, these findings further the understanding of SSRI transplacental transport and drug-induced effects on the placental barrier.

In Vitro Models for Studying Transport Across Epithelial Tissue Barriers.

Epithelial barriers are the body's natural defense system to regulating passage from one domain to another. In our efforts to understand what can and cannot cross these barriers, models have emerged as a reductionist approach to rigorously study and investigate this question. In particular, in vitro tissue models have become prominent as there is an increased exploration of understanding biological molecular transport. Herein, we introduce the pertinent physiology, then discuss recent studies and approaches for building models of five epithelial tissues: skin, the gastrointestinal tract, the lungs, the blood-brain barrier, and the placenta. In particular, we evaluated literature from the past 5 years utilizing a tissue model to evaluate molecular transport. We then compare physiology of these tissues and discuss similarities in approaches, across tissues, to validate these models. We conclude with a summary of the approaches of growing interest across multiple tissues and an outlook on future steps to improve these models.

ZEB2, a master regulator of the epithelial-mesenchymal transition, mediates trophoblast differentiation.

STUDY QUESTION: Does the upregulation of the zinc finger E-box binding homeobox 2 (ZEB2) transcription factor in human trophoblast cells lead to alterations in gene expression consistent with an epithelial-mesenchymal transition (EMT) and a consequent increase in invasiveness? SUMMARY ANSWER: Overexpression of ZEB2 results in an epithelial-mesenchymal shift in gene expression accompanied by a substantial increase in the invasive capacity of human trophoblast cells. WHAT IS KNOWN ALREADY: In-vivo results have shown that cytotrophoblast differentiation into extravillous trophoblast involves an epithelial-mesenchymal transition. The only EMT master regulatory factor which shows changes consistent with extravillous trophoblast EMT status and invasive capacity is the ZEB2 transcription factor. STUDY DESIGN, SIZE, DURATION: This study is a mechanistic investigation of the role of ZEB2 in trophoblast differentiation. We generated stable ZEB2 overexpression clones using the epithelial BeWo and JEG3 choriocarcinoma lines. Using these clones, we investigated the effects of ZEB2 overexpression on the expression of EMT-associated genes and proteins, cell morphology and invasive capability. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used lentiviral transduction to overexpress ZEB2 in BeWo and JEG3 cells. Stable clones were selected based on ZEB2 expression and morphology. A PCR array of EMT-associated genes was used to probe gene expression. Protein measurements were performed by western blotting. Gain-of-function was assessed by quantitatively measuring cell invasion rates using a Transwell assay, a 3D bioprinted placenta model and the xCelligenceTM platform. MAIN RESULTS AND THE ROLE OF CHANCE: The four selected clones (2 × BeWo, 2 × JEG3, based on ZEB2 expression and morphology) all showed gene expression changes indicative of an EMT. The two clones (1 × BeWo, 1 × JEG3) showing >40-fold increase in ZEB2 expression also displayed increased ZEB2 protein; the others, with increases in ZEB2 expression <14-fold did not. The two high ZEB2-expressing clones demonstrated robust increases in invasive capacity, as assessed by three types of invasion assay. These data identify ZEB2-mediated transcription as a key mechanism transforming the epithelial-like trophoblast into cells with a mesenchymal, invasive phenotype. LARGE SCALE DATA: PCR array data have been deposited in the GEO database under accession number GSE116532. LIMITATIONS, REASONS FOR CAUTION: These are in-vitro studies using choriocarcinoma cells and so the results should be interpreted in view of these limitations. Nevertheless, the data are consistent with in-vivo findings and are replicated in two different cell lines. WIDER IMPLICATIONS OF THE FINDINGS: The combination of these data with the in-vivo findings clearly identify ZEB2-mediated EMT as the mechanism for cytotrophoblast differentiation into extravillous trophoblast. Having characterized these cellular mechanisms, it will now be possible to identify the intracellular and extracellular regulatory components which control ZEB2 and trophoblast differentiation. It will also be possible to identify the aberrant factors which alter differentiation in invasive pathologies such as preeclampsia and abnormally invasive placenta (AKA accreta, increta, percreta). STUDY FUNDING AND COMPETING INTEREST(s): Funding was provided by the Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine and Surgery at Hackensack Meridian Health, Hackensack, NJ. The 3D bioprinted placental model work done in Drs Kim and Fisher's labs was supported by the Children's National Medical Center. The xCELLigence work done in Dr Birge's lab was supported by NIH CA165077. The authors declare no competing interests.

Biomimetic Placenta-Fetus Model Demonstrating Maternal-Fetal Transmission and Fetal Neural Toxicity of Zika Virus.

Recent global epidemics of viral infection such as Zika virus (ZIKV) and associated birth defects from maternal-fetal viral transmission highlights the critical unmet need for experimental models that adequately recapitulates the biology of the human maternal-fetal interface and downstream fetal development. Herein, we report an in vitro biomimetic placenta-fetus model of the maternal-fetal interface and downstream fetal cells. Using a tissue engineering approach, we built a 3D model incorporating placental trophoblast and endothelial cells into an extracellular matrix environment and validated formation of the maternal-fetal interface. We utilized this model to study ZIKV exposure to the placenta and neural progenitor cells. Our results indicated ZIKV infects both trophoblast and endothelial cells, leading to a higher viral load exposed to fetal cells downstream of the barrier. Viral inhibition by chloroquine reduced the amount of virus both in the placenta and transmitted to fetal cells. A sustained downstream neural cell viability in contrast to significantly reduced viability in an acellular model indicates that the placenta sequesters ZIKV consistent with clinical observations. These findings suggest that the placenta can modulate ZIKV exposure-induced fetal damage. Moreover, such tissue models can enable rigorous assessment of potential therapeutics for maternal-fetal medicine.

Mechanical fractionation of tissues using microsecond-long HIFU pulses on a clinical MR-HIFU system.

PURPOSE: High intensity focussed ultrasound (HIFU) can non-invasively treat tumours with minimal or no damage to intervening tissues. While continuous-wave HIFU thermally ablates target tissue, the effect of hundreds of microsecond-long pulsed sonications is examined in this work. The objective of this study was to characterise sonication parameter-dependent thermomechanical bioeffects to provide the foundation for future preclinical studies and facilitate clinical translation. METHODS AND MATERIALS: Acoustic power, number of cycles/pulse, sonication time and pulse repetition frequency (PRF) were varied on a clinical magnetic resonance imaging (MRI)-guided HIFU (MR-HIFU) system. Ex vivo porcine liver, kidney and cardiac muscle tissue samples were sonicated (3 × 3 grid pattern, 1 mm spacing). Temperature, thermal dose and T2 relaxation times were quantified using MRI. Lesions were histologically analysed using H&E and vimentin stains for lesion structure and viability. RESULTS: Thermomechanical HIFU bioeffects produced distinct types of fractionated tissue lesions: solid/thermal, paste-like and vacuolated. Sonications at 20 or 60 Hz PRF generated substantial tissue damage beyond the focal region, with reduced viability on vimentin staining, whereas H&E staining indicated intact tissue. Same sonication parameters produced dissimilar lesions in different tissue types, while significant differences in temperature, thermal dose and T2 were observed between the parameter sets. CONCLUSION: Clinical MR-HIFU system was utilised to generate distinct types of lesions and to produce targeted thermomechanical bioeffects in ex vivo tissues. The results guide HIFU research on thermomechanical tissue bioeffects, inform future studies and advice sonication parameter selection for direct tumour ablation or immunomodulation using a clinical MR-HIFU system.

Magnetic Resonance Imaging-guided High-intensity Focused Ultrasound Applications in Pediatrics: Early Experience at Children's National Medical Center.

Magnetic resonance imaging-guided high-intensity focused ultrasound (MR-HIFU) is a novel technology that integrates magnetic resonance imaging with therapeutic ultrasound. This unique approach provides a completely noninvasive method for precise thermal ablation of targeted tissues with real-time imaging feedback. Over the past 2 decades, MR-HIFU has shown clinical success in several adult applications ranging from treatment of painful bone metastases to uterine fibroids to prostate cancer and essential tremor. Although clinical experience in pediatrics is relatively small, the advantages of a completely noninvasive and radiation-free therapy are especially attractive to growing children. Unlike elderly patients, young children must deal with an entire lifetime of negative effects related to collateral tissue damage associated with invasive surgery, side effects of chemotherapy, and risk of secondary malignancy due to radiation exposure. These reasons provide a clear rationale and strong motivation to further advance clinical utility of MR-HIFU in pediatrics. We begin with an introduction to MR-HIFU technology and the clinical experience in adults. We then describe our early institutional experience in using MR-HIFU ablation to treat symptomatic benign, locally aggressive, and metastatic tumors in children and young adults. We also review some limitations and challenges encountered in treating pediatric patients and highlight additional pediatric applications which may be feasible in the near future.