Intra-amniotic sildenafil administration in rabbits: Safety, pharmacokinetics, organ distribution and histologic evaluation.

BACKGROUND: The effectiveness of sildenafil in the management of pulmonary hypertension in congenital diaphragmatic hernia (CDH) has been reported but has not been systematically evaluated. Our studies have also demonstrated that intra-amniotic (IA) sildenafil administration improves pulmonary hypertension in CDH. METHODS: We evaluated the pharmacokinetics of sildenafil after IA administration in pregnant rabbits. Following maternal laparotomy, fetuses received IA injection of 0.8 mg of sildenafil. Maternal blood, amniotic fluid, and fetal tissues were collected at various time points. The concentrations of sildenafil and its major metabolite in samples were analyzed by liquid chromatography-mass spectrometry. To assess organ toxicity, 7 days after IA sildenafil administration, fetal organs were examined histologically. RESULTS: After IA dosing, sildenafil was absorbed quickly with an absorption half-life of 0.03-0.07 h into the fetal organs. All the organs showed a maximum concentration within 1 h and the disposition half-life ranged from 0.56 to 0.73 h. Most of the sildenafil was eliminated from both mothers and fetuses within 24 h after a single dose. There was no histological evidence of organ toxicity in the fetuses after a single dose of IA administration of sildenafil. CONCLUSION: IA sildenafil is rapidly absorbed into the fetus, distributes into the mother, and is eliminated by the mother without accumulation or fetal organ toxicity. This study confirms the feasibility and the safety of IA administration of sildenafil and enables future applications in the treatment of CDH fetuses.

Comparison of surgical outcomes between laparoscopic percutaneous extracorporeal closure (LPEC) and open repair for pediatric inguinal hernia by propensity score methods and log-rank test analysis.

BACKGROUND: Despite a number of studies comparing laparoscopic inguinal hernia repair (LH) and open herniorrhaphy (OH), the putative advantage of LH remains controversial due to a paucity of firm evidence. We hypothesized that LH has both advantages and disadvantages compared to OH and sought to clarify them by comprehensively analyzing the retrospective data using the combination of multiple statistical methods. METHODS: Operative data for inguinal hernia during the period from February 1999 to December 2019 were examined. The patients were assigned into two groups according to the surgical procedure: laparoscopic percutaneous extraperitoneal closure (LPEC, n = 2410) and OH (n = 2038). Operative and anesthesia times and incidence of postoperative complications were evaluated using the propensity score methods and log-rank test. RESULTS: In comparison with OH, operative time of LPEC was longer for unilateral repair (21.59 ± 8.1 min vs 18.01 ± 8.0 min; p < 0.001) and shorter for bilateral repairs (28.55 ± 10.1 min vs 33.23 ± 11.7 min; p < 0.001), while anesthesia times were longer for both unilateral repair (57.67 ± 10.1 min vs 40.62 ± 11.9 min; p < 0.001) and bilateral repairs (65.95 ± 12.5 min vs 56.35 ± 15.1 min; p < 0.001). LPEC significantly reduced the risk of metachronous contralateral hernia (MCLH) (0.52% vs 9.29%; p < 0.001), but the recurrence rate was higher (0.21% vs 0.04%; p = 0.002) than OH. Orchiectomy due to testicular atrophy or torsion was required in 3 cases of OH (0.19%), whereas it was not seen in LPEC. CONCLUSIONS: LPEC had a less risk of MCLH and testicular complications but was associated with a higher recurrence rate and longer anesthesia time. Propensity scoring techniques can enhance the robustness of retrospective comparisons between groups over several years of data collection, which is frequently required in pediatric surgery studies.

Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens.

PURPOSE: Refinement of organoid technology is important for studying physiology and disease of the intestine. We aimed to optimize defined serum-free conditions for human infant small intestinal (SI) organoid culture with predetermined doses of Wnt3a and Rspo1 from surgical specimens. We further assessed whether intestinal specimens could be stored before use as a source of organoids. METHODS: Different doses of Wnt3a and Rspo1 in a serum-free medium were tested to establish a condition in which surgically resected SI cells grew as organoids over multiple passages. The expression of marker genes for stem and differentiated cells was assessed by quantitative polymerase chain reaction. We also investigated the organoid-forming efficiency of cells in degenerating intestines stored at 4 °C for various intervals post-resection. RESULTS: We determined the doses of Wnt3a and Rspo1 required for the continuous growth of infant SI organoids with multi-differentiation potential. We revealed that, despite the time-dependent loss of stem cells, tissues stored for up to 2 days preserved cells capable of generating amplifiable organoids. CONCLUSION: SI cells can be grown as organoids under defined conditions. This could provide a reproducible and customizable method of using surgical specimens for the study of intestinal maturation and their relevance to pediatric diseases.

Perforation in pediatric non-complicated appendicitis treated by antibiotics: the real incidence.

PURPOSE: The incidence of perforation during antibiotic therapy (AT) of children triaged as non-complicated acute appendicitis (NC-Ap) was investigated. METHODS: Abdominal ultrasonography (US) and/or computed tomography (CT) scans from cases of perforation identified at appendectomy for failed AT were reassessed blindly by a panel of board-certified specialists for any evidence of pre-AT morbidity suggestive of perforation. RESULTS: Of 521 cases triaged as NC-Ap, symptoms resolved with AT in 452 cases (86.8%). All 69/521 (13.2%) cases with persistent symptoms had urgent appendectomy, and 12/521 (2.3%) were found to have perforated. Blind reassessment of US and/or CT scans from these cases identified seven with evidence of perforation when they were triaged as NC-Ap. Thus, the actual incidence of perforation during AT for NC-Ap was actually 12-7 = 5/521 (0.95%). CONCLUSIONS: Perforation is generally believed to be a complication of AT, but inappropriate triaging of cases for AT can bias results by artificially inflating the number of perforations, in this study, by more than double. We are the first to assess the unbiased incidence of perforation during AT for NC-Ap, by reassessing pre-AT US and/or CT scans. The incidence of perforation during AT is actually negligible.

Pregnane X receptor and hepatocyte nuclear factor 4α polymorphisms are cooperatively associated with carbamazepine autoinduction.

OBJECTIVE: We attempted to clarify the influence of polymorphisms of nuclear receptors on carbamazepine therapy. PARTICIPANTS AND METHODS: The common polymorphisms of nuclear receptors--a tentative pregnane X receptor (PXR)*1B, hepatocyte nuclear factor 4α (HNF4α) rs2071197 (c.115+308G>A), and cytochrome P450 3A5*3 polymorphisms--were genotyped in 168 Japanese patients with epilepsy. The associations of these polymorphisms with the disposition, clinical efficacy, and incidence of adverse effects of carbamazepine treatment were retrospectively investigated in 104 patients treated with carbamazepine alone. The associations with disposition were also assessed in 64 patients treated with carbamazepine and other antiepileptic drugs, which constituted the internal replication group, and in the combined 168 patients. RESULTS: Neither polymorphism alone affected the carbamazepine disposition, but a significant interactive effect of PXR*1B and HNF4α rs2071197 polymorphisms on the concentration-to-dose (C/D) ratios was observed (P=0.027). The C/D ratios among patients with the HNF4α G/G genotype were higher in PXR*1B carriers than in PXR*1B noncarriers, which was confirmed in the internal replication and combined groups. In patients with the HNF4α G/G genotype, the rate of freedom from seizures until 3 months after starting carbamazepine therapy was significantly greater and the time required to reach the dose required for seizure freedom was shorter in PXR*1B carriers than in PXR*1B noncarriers. CONCLUSION: These results suggest that PXR*1B, in combination with HNF4α rs2071197, might be associated with the C/D ratios and the duration to reach the maintenance dose of carbamazepine therapy, thus indicating an influence upon the autoinduction of the carbamazepine metabolism.

Intra-Amniotic Sildenafil and Rosiglitazone Late in Gestation Ameliorate the Pulmonary Hypertension Phenotype in Congenital Diaphragmatic Hernia.

BACKGROUND: Pulmonary hypertension remains difficult to manage in congenital diaphragmatic hernia (CDH). Prenatal therapy may ameliorate postnatal pulmonary hypertension. We hypothesized that intra-amniotic (IA) injection of either sildenafil, a phosphodiesterase 5 inhibitor, or rosiglitazone, a PPAR-γ agonist, or both late in gestation would decrease the detrimental pulmonary vascular remodeling seen in CDH and improve peripheral pulmonary blood flow. METHODS: Pregnant rats were gavaged with nitrogen on embryonic day (E) 9.5 to induce fetal CDH. Sildenafil and/or rosiglitazone were administered to each fetus via an intra-amniotic injection after laparotomy on the pregnant dam at E19.5, and fetuses delivered at E21.5. Efficacy measures were gross necropsy, histology, peripheral blood flow assessment using intra-cardiac injection of a vascular tracer after delivery, and protein expression analysis. RESULTS: Intra-amniotic injections did not affect fetal survival, the incidence of CDH, or lung weight-to-body weight ratio in CDH fetuses. IA sildenafil injection decreased pulmonary vascular muscularization, and rosiglitazone produced an increase in peripheral pulmonary blood flow distribution. The combination of sildenafil and rosiglitazone decreased pulmonary artery smooth muscle cell proliferation. These intra-amniotic treatments did not show any negative effects in either CDH fetuses or control fetuses. CONCLUSION: IA injection of sildenafil and rosiglitazone late in gestation ameliorates the pulmonary hypertensive phenotype of CDH and may have utility in clinical translation. LEVEL OF EVIDENCE: Not applicable.

Acinar to ß-like cell conversion through inhibition of focal adhesion kinase.

Insufficient functional ß-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing ß-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore ß-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes.

Phase I/II clinical trial of brentuximab vedotin for pretreated Japanese patients with CD30-positive cutaneous T-cell lymphoma.

Brentuximab vedotin (BV), a conjugate of anti-CD30 antibody and monomethyl auristatin E, has emerged as a promising treatment option for refractory CD30+ mycosis fungoides (MF) and primary cutaneous anaplastic large-cell lymphoma (pcALCL). BV has been shown to be safe and effective in treating Hodgkin's lymphoma and peripheral T-cell lymphoma. This multicenter, prospective, single-arm phase I/II study evaluated the efficacy of BV in Japanese patients with CD30+ cutaneous lymphomas, namely CD30+ cutaneous T-cell lymphoma. Participants were divided into two groups: those with CD30+ MF or pcALCL (cohort 1, n = 13) and those with CD30+ lymphoproliferative disorders other than those in cohort 1 (cohort 2, n = 3). The studied population included the full analysis set (FAS), modified FAS (mFAS), and safety analysis set (SAF). These sets were identified in cohorts 1 and 1 + 2 and labeled FAS1 and FAS2, mFAS1 and mFAS2, and SAF1 and SAF2, respectively. Each treatment cycle lasted 3 weeks, and BV was continued for up to 16 cycles after the third cycle based on treatment response. The primary endpoint was the 4-month objective response rate (ORR4) determined by the Independent Review Forum (IRF). ORR4 was 69.2% for FAS1 and 62.5% for FAS2 (P < 0.0001). Secondary endpoints of ORR, assessed using the global response score (53.8% in FAS1) and modified severity-weighted assessment tool (62.5% in FAS1), using the IRF, provided results comparable to the primary findings. The incidence of ≥grade 3 adverse events (≥15%) in SAF1 was peripheral neuropathy in three patients (23%) and fever and eosinophilia in two patients (15%). In conclusion, BV showed favorable efficacy, tolerability, and safety profile in Japanese patients with relapsed or refractory CD30+ primary cutaneous T-cell lymphoma. The trial was registered with University Hospital Medical Information Network Clinical Trials Registry, Japan (protocol ID: UMIN000034205).

Intra-amniotic sildenafil treatment improves lung blood flow and pulmonary hypertension in congenital diaphragmatic hernia rats.

Pulmonary hypertension associated with congenital diaphragmatic hernia (CDH) is a critical factor in determining prognosis. We propose that intra-amniotic sildenafil administration is an effective prenatal therapy for CDH-induced pulmonary hypertension. To assess the efficacy of this treatment, we administered sildenafil to nitrofen-induced congenital diaphragmatic hernia fetuses and control fetuses via an intra-amniotic injection after a laparotomy on the pregnant dam at either E13.5 or E15.5. Intra-amniotic sildenafil treatment attenuated peripheral vascular muscularization, enhanced pulmonary blood flow, and increased the ratio of pulmonary artery size to aortic size in congenital diaphragmatic hernia fetuses after both E13.5 and E15.5 treatments. E13.5-treated congenital diaphragmatic hernia fetuses showed a higher and more prolonged expression of cyclic guanosine monophosphate (cGMP)-dependent protein kinase and more production of vascular endothelial growth factor, resulting in a significant improvement in lung architecture. The E13.5-treated congenital diaphragmatic hernia fetuses also had an increase in lung weight-to-body weight ratio and an improved fetal survival. Intra-amniotic sildenafil treatment did not show any detectable negative effects in control fetuses. Intra-amniotic sildenafil treatment for rats attenuates CDH-induced pulmonary hypertension and enhanced peripheral pulmonary blood flow. Moreover, early intervention may be preferable to better accelerate lung development and improve prognosis. Direct sildenafil administration via an intra-amniotic injection may be a promising option in congenital diaphragmatic hernia prenatal therapy.

Successful all robotic-assisted excision of highly malignant mediastinal neuroblastoma in a toddler: A case report.

An otherwise well 28-month-old girl presented with fever/left thigh pain. Computed tomography identified a 7 cm right posterior mediastinal tumor extending to the paravertebral and intercostal spaces with multiple bone and bone marrow metastases on bone scintigraphy. Thoracoscopic biopsy diagnosed MYCN non-amplified neuroblastoma. Chemotherapy shrank the tumor to 5 cm by 35 months of age. Robotic-assisted resection was chosen because the patient was large enough and public health insurance coverage was available. At surgery, the tumor was well-demarcated by chemotherapy and dissection posteriorly from the ribs/intercostal spaces and medially from the paravertebral space and azygos vein was facilitated by superior visualization/instrument articulation. The capsule of the resected specimen was intact on histopathology, confirming complete tumor resection. Despite minimum distance specifications between arms, trocars, and target sites with robotic assistance, excision was safe without instrument collisions. Robotic assistance should be actively considered for pediatric malignant mediastinal tumor provided the thorax is of adequate size.

Remote cadaveric minimally invasive surgical training.

Objective: The aim of the study is to discuss the efficacy of live vs. remote cadaver surgical training (CST) for minimally invasive surgery (MIS). Methods: A cohort of 30 interns in their first and second years of training were divided into three groups: live observers (n = 12), live participants (n = 6), and remote observers: (n = 12). The interns had the opportunity to either observe or actively participate in two different surgical procedures, namely, laparoscopic lower anterior resection, performed by a colorectal surgical team, and laparoscopic fundoplication, performed by a pediatric surgical team. The procedures were conducted either at a base center or at a remote center affiliated with the institute. Some of the interns interacted directly with the surgical teams at the base center, and others interacted indirectly with the surgical teams from the remote center. All interns were administered questionnaires before and after completion of the CST in order to assess their understanding of various aspects related to the operating room layout/instruments (called "design"), accessing the surgical field (called "field"), understanding of anatomic relations (called "anatomy"), their skill of dissection (called "dissection"), ability to resolve procedural/technical problems (called "troubleshooting"), and their skill in planning surgery (called "planning") according to their confidence to operate using the following scale: 1 = not confident to operate independently; 4 = confident to operate with a more senior trainee; 7 = confident to operate with a peer; and 10 = confident to operate with a less experienced trainee. A p < 0.05 was considered statistically significant. Results: All scores improved after CST at both the base and remote centers. The following significant increases were observed: for remote observers: "field" (2.67→4.92; p < .01), "anatomy" (3.58→5.75; p < .01), "dissection" (3.08→4.33; p = .01), and "planning" (3.08→4.33; p < .01); for live observers: "design" (3.75→6.17; p < .01), "field" (2.83→5.17; p < .01), "anatomy" (3.67→5.58; p < .01), "dissection" (3.17→4.58; p < .01), "troubleshooting" (2.33→3.67; p < .01), and "planning" (2.92→4.25; p < .01); and for live participants: "design" (3.83→6.33; p = .02), "field" (2.83→6.83; p < .01), "anatomy" (3.67→5.67; p < .01), "dissection" (2.83→6.17; p < .01), "troubleshooting" (2.17→4.17; p < .01), and "planning" (2.83→4.67; p < .01). Understanding of "design" improved significantly after CST in live observers compared with remote observers (p < .01). Understanding of "field and "dissection" improved significantly after CST in live participants compared with live observers (p = .01, p = .03, respectively). Out of the 12 remote observers, 10 participants (83.3%) reported that interacting with surgical teams was easy because they were not on-site. Conclusions: Although all the responses were subjective and the respondents were aware that observation was inferior to hands-on experience, the results from both centers were equivalent, suggesting that remote learning could potentially be viable when resources are limited.

Chemical pancreatectomy in non-human primates ablates the acini and ducts and enhances beta-cell function.

Chronic pancreatitis is a debilitating disease affecting millions worldwide. These patients suffer from bouts of severe pain that are minimally relieved by pain medications and may necessitate major surgeries with high morbidity and mortality. Previously, we demonstrated that "chemical pancreatectomy," a pancreatic intraductal infusion of dilute acetic acid solution, ablated the exocrine pancreas while preserving the endocrine pancreas. Notably, chemical pancreatectomy resolved chronic inflammation, alleviated allodynia in the cerulein pancreatitis model, and improved glucose homeostasis. Herein, we extensively tested the feasibility of a chemical pancreatectomy in NHPs and validated our previously published pilot study. We did serial computed tomography (CT) scans of the abdomen and pelvis, analyzed dorsal root ganglia, measured serum enzymes, and performed histological and ultrastructural assessments and pancreatic endocrine function assays.  Based on serial CT scans, chemical pancreatectomy led to the loss of pancreatic volume. Immunohistochemistry and transmission electron microscopy demonstrated exocrine pancreatic ablation with endocrine islet preservation. Importantly, chemical pancreatectomy did not increase pro-nociceptive markers in harvested dorsal root ganglia. Also, chemical pancreatectomy improved insulin secretion to supranormal levels in vivo and in vitro. Thus, this study may provide a foundation for translating this procedure to patients with chronic pancreatitis or other conditions requiring a pancreatectomy.

Chemical pancreatectomy in non-human primates ablates the acini and ducts and enhances beta-cell function.

Chronic pancreatitis is a debilitating disease affecting millions worldwide. These patients suffer from bouts of severe pain that are minimally relieved by pain medications and may necessitate major surgeries with high morbidity and mortality. Previously, we demonstrated that "chemical pancreatectomy," a pancreatic intraductal infusion of dilute acetic acid solution, ablated the exocrine pancreas while preserving the endocrine pancreas. Notably, chemical pancreatectomy resolved chronic inflammation, alleviated allodynia in the cerulein pancreatitis model, and improved glucose homeostasis. Herein, we extensively tested the feasibility of a chemical pancreatectomy in NHPs and validated our previously published pilot study. We did serial computed tomography (CT) scans of the abdomen and pelvis, analyzed dorsal root ganglia, measured serum enzymes, and performed histological and ultrastructural assessments and pancreatic endocrine function assays. Based on serial CT scans, chemical pancreatectomy led to the loss of pancreatic volume. Immunohistochemistry and transmission electron microscopy demonstrated exocrine pancreatic ablation with endocrine islet preservation. Importantly, chemical pancreatectomy did not increase pro-nociceptive markers in harvested dorsal root ganglia. Also, chemical pancreatectomy improved insulin secretion to supranormal levels in vivo and in vitro. Thus, this study may provide a foundation for translating this procedure to patients with chronic pancreatitis or other conditions requiring a pancreatectomy.

Successful closure of a large abdominal wall defect using endoscopic component separation technique in an infant with a giant ventral hernia after staged surgery for omphalocele.

BACKGROUND: The management of large abdominal wall defects, such as omphalocele or gastroschisis, remains a challenge for pediatric surgeons. Though several techniques have been described to repair those conditions, there is no procedure considered to be the standard worldwide. We report an infant girl with a giant ventral hernia after staged surgery for omphalocele in whom delayed closure of a large abdominal wall defect was performed successfully using "endoscopic component separation technique (ECST)" without serious complications. CASE PRESENTATION: A baby girl was admitted to our hospital because of a giant omphalocele, which had been prenatally diagnosed. The omphalocele was supraumbilical and included the entire liver. After staged surgery, a large abdominal wall defect was closed by skin, creating a giant ventral hernia. We performed endoscopic separation component technique (ECST) for the closure of her abdominal wall defect when she was 11 months of age. ECST was initiated with placement of a 5.0-mm port just above the inguinal ligament and under the external oblique muscle. The space between the external and internal oblique muscles was created by the insufflation pressure, and a second 5.0-mm port was placed at 1.0 cm below the inferior edge of the rib into the space. As the further dissection was carried, the aponeurosis of the external oblique muscle was identified as a white line, running vertically from the epigastrium to inguinal ligament. It was transected longitudinally using electrocautery over its full length. The same procedure was performed on the contralateral side and the abdominal wall was successfully closed. Postoperative course was uneventful. CONCLUSIONS: The technique of ECST, described here, is simple and safe for infants, and the cosmetic result is satisfactory.

ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein.

Objective: Acrolein is a highly reactive aldehyde that covalently binds to cellular macromolecules and subsequently modulates cellular function. Our previous study demonstrated that acrolein induces glial cell migration, a pathological hallmark of diabetic retinopathy; however, the detailed cellular mechanism remains unclear. The purpose of this study was to investigate the role of acrolein in retinal glial cell migration by focusing on rho-associated coiled-coil-containing protein kinases (ROCKs). Methods: Immunofluorescence staining for ROCK isoforms was performed using sections of fibrovascular tissue obtained from the eyes of patients with proliferative diabetic retinopathy (PDR). Rat retinal Müller glial cell line, TR-MUL5, was stimulated with acrolein and the levels of ROCK1 were evaluated using real-time PCR and western blotting. Phosphorylation of the myosin-binding subunit of myosin light chain phosphatase [myosin phosphatase target subunit 1, (MYPT1)] and myosin light chain 2 (MLC2) was assessed. The cell migration rate of TR-MUL5 cells exposed to acrolein and/or ripasudil, a non-selective ROCK inhibitor, was measured using the Oris cell migration assay. Results: ROCK isoforms, ROCK1 and ROCK2, were positively stained in the cytosol of glial cells in fibrovascular tissues. In TR-MUL5 cells, the mRNA expression level of Rock1, but not Rock2, was increased following acrolein stimulation. In line with the PCR data, western blotting showed increase in ROCK1 and cleaved ROCK1 protein in TR-MUL5 cells stimulated with acrolein. N-acetylcysteine (NAC) suppressed acrolein-associated Rock1 upregulation in TR-MUL5 cells. Acrolein augmented the phosphorylation of MYPT1 and MLC2 and increased the cell migration rate of TR-MUL5 cells, both of which were abrogated by ripasudil. Conclusions: Our study demonstrated that ROCK1 mediates the migration of retinal glial cells promoted by the unsaturated aldehyde acrolein.

Cadaver Training for Minimally Invasive Pediatric Surgery: A Preliminary Report.

Aim: To report the value of adult cadavers for training in minimally invasive surgical (MIS) techniques for pediatric surgery (PS). Materials and Methods: Three teams, each consisting of a board-certified consultant pediatric surgeon (CS), a senior trainee (ST), and a junior trainee (JT), attended a cadaver surgical training (CST) course involving five procedures: thoracoscopic esophagoesophagostomy (TEE), thoracoscopic right lower lobectomy (TRL), laparoscopic fundoplication (LFN), laparoscopic hepaticoduodenostomy (LHD), and laparoscopic ureteroureterostomy (LUU). The same teams also performed LFN on live pigs. Attendees (3 CSs, 3 STs, and 3 JTs) were administered a questionnaire to rate their CST experience according to five criteria (tissue texture, organ size, operative field, "feel," and anatomic relationships) using a 4-point scale with 0 being the worst response. Scores were averaged per procedure per attendee groups and compared. LFN was also compared between a cadaver and a pig. Results: End-point (1): For LFN, cadaver scores were significantly higher than pig scores for anatomic relationships (P = .0001), operative field (P = .0053), and organ size (P = .0481). End-point (2): TRL and LFN were ranked together as being most realistic, followed by TEE and LUU, then LHD. End-point (3): Anatomic relationships and operative field consistently scored highly for all attendee groups. End-point (4): CSs and STs tended to award higher scores than JTs although differences were not statistically significant. Conclusions: CST is a valuable opportunity for PS trainees to experience highly realistic training in minimally invasive surgery. Pig training was inferior. IRB Number: 2019173.

Unsaturated Aldehyde Acrolein Promotes Retinal Glial Cell Migration.

Purpose: To investigate the effect of the unsaturated aldehyde acrolein on retinal glial cell migration. Methods: Müller glial cell markers expression in TR-MUL5 were confirmed by RT-PCR and immunostaining. Cell viability and migration rate of TR-MUL5 cells were assessed after the stimulation with acrolein. DNA microarray analysis was performed to analyze changes in the expression levels of migration-related genes in Müller glial cells stimulated with acrolein. Real-time PCR and ELISA were performed to validate DNA microarray analysis results. Inhibitors of C-X-C motif chemokine ligand 1 (CXCL1), one of the genes highly upregulated after the exposure to acrolein, and blockers of its receptor, CXCR2, were used to investigate the role of the CXCL1-CXCR2 axis on glial cell migration. CXCL1 concentration was measured in vitreous fluid samples obtained from proliferative diabetic retinopathy (PDR) and nondiabetic control eyes. CXCL1 and CXCR2 expression in glial cells of fibrovascular tissues obtained from PDR patients was examined by immunostaining. Results: At a high concentration, acrolein (100 µM) significantly decreased cell viability. However, in moderate, sublethal concentrations (25-50 µM), acrolein induced cell migration and substantially increased the production of CXCL1 in TR-MUL5 cells. CXCL1 concentration was significantly elevated in vitreous fluids of PDR patients, and CXCL1 and CXCR2 were present in glial cells in fibrovascular tissues of PDR patients. CXCL1 stimulation increased glial cell migration in a dose-dependent manner, which was abrogated by the neutralization of the CXCL1-CXCR2 axis. Conclusions: Our data demonstrate that acrolein promotes retinal Müller glial cell migration by enhancing CXCL1 production.

Proteolytic cleavage of vascular adhesion protein-1 induced by vascular endothelial growth factor in retinal capillary endothelial cells.

PURPOSE: To investigate the mechanism of soluble vascular adhesion protein-1 (sVAP-1) accumulation induced by vascular endothelial growth factor (VEGF) in the vitreous of patients with diabetic retinopathy (DR). STUDY DESIGN: Experimental. METHODS: Protein levels of sVAP-1 and N epsilon-(hexanoyl)lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with proliferative diabetic retinopathy (PDR) with or without intravitreal bevacizumab (IVB) injection were determined by ELISA. The effect of VEGF on both mRNA expression of Vap-1 and secretion of sVAP-1 in rat retinal capillary endothelial cells (TR-iBRB2) was analyzed by real-time PCR and western blotting, respectively. In addition, the impact of VEGF on production and activation ratios of matrix metalloproteinase (MMP)-2 and MMP-9 was examined by gelatin zymography. Hydrogen peroxide production and reactive oxygen species (ROS) levels were assessed in the supernatants of TR-iBRB2 cells treated with VEGF. RESULTS: IVB injection decreased vitreous levels of sVAP-1 and HEL in patients with PDR. VEGF stimulation released sVAP-1 protein from TR-iBRB2 cells as a consequence of membrane-anchored VAP-1 shedding by MMP-2 and MMP-9. In addition, VEGF increased hydrogen peroxide generation and ROS augmentation through spermine oxidation by sVAP-1 as semicarbazide-sensitive amine oxidase (SSAO) in the supernatant of cultured endothelial cells. CONCLUSIONS: The current data demonstrate that proangiogenic factor VEGF induces sVAP-1 release from retinal capillary endothelial cells and facilitates hydrogen peroxide generation via enzymatic property of sVAP-1, followed by the increase of oxidative stress, one of the crucial factors in the pathogenesis of DR.

Localization of Acrolein-Lysine Adduct in Fibrovascular Tissues of Proliferative Diabetic Retinopathy.

PURPOSE: To determine the presence of Nε-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), unsaturated aldehyde acrolein-derived lipoxidation end-product, in fibrovascular tissues obtained from patients with proliferative diabetic retinopathy (PDR). METHODS: Fibrovascular tissues were collected from 11 eyes of 11 patients with PDR and paraffin-embedded tissue sections were prepared. Tissue localization of FDP-Lys was studied by immunohistochemistry. Signal intensity was quantified by two masked evaluators and graded into three discrete categories. The relationship between FDP-Lys staining and vascular density was analyzed. In addition, subcellular localization of FDP-Lys was studied by immunofluorescent microscopy. The impact of acrolein on cell viability and proliferation was assessed and the expression level of heme oxygenase-1 (HO-1) mRNA was quantified by real-time polymerase chain reaction (PCR) in cultured retinal microvascular endothelial cells. RESULTS: In fibrovascular tissues, FDP-Lys staining was found in vascular components containing CD34-positive cells and alpha smooth muscle actin (α-SMA)-positive cells, and clusters of rabbit anti-glial fibrillary acid protein (GFAP)-positive cells. Immunofluorescent staining depicted subcellular localization of FDP-Lys in the nucleus and cytoplasm of the cells. Morphological analysis revealed that fibrovascular tissues with FDP-Lys staining in vascular components showed high vascular density. Exposure of cultured endothelial cells to high concentration of acrolein resulted in the decrease of cell viability and proliferation, whereas lower concentration of acrolein increased cell viability and proliferation. Sublethal concentration of acrolein upregulated HO-1 mRNA expression in retinal microvascular endothelial cells. CONCLUSIONS: The current data demonstrated the presence of FDP-Lys in fibrovascular tissues and indicate its involvement in fibrovascular proliferation in PDR.

Soluble Vascular Adhesion Protein-1 Mediates Spermine Oxidation as Semicarbazide-Sensitive Amine Oxidase: Possible Role in Proliferative Diabetic Retinopathy.

Purpose/Aim of the study: To explore the possible role of vascular adhesion protein-1 (VAP-1) via its enzymatic function as a semicarbazide-sensitive amine oxidase (SSAO) in the pathogenesis of proliferative diabetic retinopathy (PDR). MATERIALS AND METHODS: The levels of soluble VAP-1/SSAO and the unsaturated aldehyde acrolein (ACR)-conjugated protein, Nε-(3-formyl-3, 4-dehydropiperidino) lysine adduct (FDP-Lys), were measured in vitreous fluid samples of PDR and non-diabetic patients using ELISA. Recombinant human VAP-1/SSAO (rhVAP-1/SSAO) was incubated with spermine, with or without semicarbazide or RTU-1096 (a specific inhibitor for VAP-1/SSAO). Immunofluorescence assays were performed to assess the localization of VAP-1/SSAO and FDP-Lys in fibrovascular tissues from patients with PDR. The impact of ACR on cultured retinal capillary endothelial cells was assessed using a cell viability assay and total glutathione (GSH) measurements. RESULTS: The levels of sVAP-1/SSAO and FDP-Lys were elevated in the vitreous fluid of patients with PDR. Incubation of rhVAP-1 with spermine resulted in the generation of hydrogen peroxide and FDP-Lys and the production was inhibited by semicarbazide and RTU-1096. In fibrovascular tissues, FDP-Lys and VAP-1/SSAO were present in endothelial cells. ACR stimulation reduced GSH levels in the cultured endothelial cells in a dose-dependent manner and caused cellular toxicity. CONCLUSIONS: Our results indicate the pathological role of sVAP-1/SSAO to generate hydrogen peroxide and toxic aldehyde ACR, both of which are associated with oxidative stress, as a consequence of spermine oxidation in eyes with PDR.