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The fabrication of SrBi[sub 2]Ta[sub 2]O[sub 9] (SBT) films using plasma-assisted metalorganic chemical vapor deposition (P-MOCVD) has been investigated. Optimizing the process conditions under plasma environment, amorphous SBT films were successfully deposited at a substrate temperature below 300 °C, suggesting that the P-MOCVD process effectively utilizes plasma energy to promote the reaction and decomposition of metal organic source molecules. The amorphous SBT films were crystallized to the bilayered perovskite SBT films by a postannealing at 725 °C. Thin SBT capacitors fabricated using P-MOCVD showed a good step coverage and the excellent ferroelectric properties including endurance. Low voltage operation below 1.5 V was successfully achieved using a 75 nm SBT capacitor, in which the signal level derived from the hysteresis curve suggests the feasibility of application to a 64 Mbit ferroelectric random access memories. © 2001 American Institute of Phys! ics. [ABSTRACT FROM AUTHOR]

Background A merit of subnormothermic perfusion has been reported to preserve grafts from ischemic injury in animal models. The split liver technique is commonly performed to solve the shortage of liver grafts. However, there has been no study showing the effect of a split liver graft on subnormothermic perfusion. We herein investigated the split liver protocol using a subnormothermic oxygenated circuit system (SOCS). Methods Auxiliary liver transplantation was performed in a porcine marginal donor model by using a SOCS. In the SOCS group, the portal vein and hepatic artery of the graft were cannulated, and the graft was perfused by SOCS. In the cold storage (CS) group, the graft was placed in cold preservation solution. In the preservation phase, the graft was split. Results There were no significant differences in the biochemical markers between the SOCS and CS groups. In terms of the histology, the sinusoidal spaces were widened in the CS group 12 hours after implantation. Conclusion We have demonstrated a possibility to use SOCS with the split liver protocol by using a porcine model. This split liver protocol using SOCS will extend the split liver criteria and rescue more patients from hepatic failure, including pediatric patients. [ABSTRACT FROM AUTHOR]

Abstract: Background: Acute cellular rejection (ACR) is a common cause of morbidity following liver transplantation. Several reports have evaluated the predictive value of peripheral blood eosinophilia as a simple noninvasive diagnostic marker for ACR. This study examined whether the relative eosinophil counts (REC) predicted ACR in pediatric living donor liver transplantation (LDLT). Methods: One hundred three patients underwent LDLT between May 2001 and December 2007. ACR were diagnosed based on the pathological findings. Results: The incidence of ACR was 46.6% (48/103); ACR was diagnosed an average of 13.5 days after LDLT. The average REC at 4 and 2 days before the onset ACR (n = 39) within 30 postoperative day (POD) was 4.3% and 7.3%, respectively, and 9.0% at the onset. Patients with ACR showed significantly higher levels of REC compared with those free of ACR (P = .039). REC thresholds of 10% at POD 7 displayed a sensitivity and specificity of ACR detection of 80% and 75%, respectively. Moreover, the accumulated morbidity ratio of ACR within 30 POD was significantly higher with REC >10% at POD 7 (P = .007). Conclusion: ACR within POD 30 should be considered when REC is >10% at POD 7 after LDLT. [Copyright &y& Elsevier]

Abstract: Objectives: Cholestatic liver disease (CLD) is the main indication for liver transplantation in children. This retrospective study evaluated the outcomes of living donor liver transplantation (LDLT) in children with CLD. Methods: One hundred fifty-nine children with CLD who underwent 164 LDLT between May 2001 and May 2011 were evaluated. Their original diseases were biliary atresia (n = 145, 91%), Alagille syndrome (n = 8, 5%), primary sclerosing cholangitis (n = 2), and the others (n = 4). The mean age and body weight of the recipients at LDLT was 42 ± 53 months and 14.0 ± 11.0 kg, respectively. Results: Parents were living donors in 98%. The left lateral segment was the most common type of graft (77%). There were no reoperations and no mortality in any living donor. Recipients'' postoperative surgical complications consisted mainly of hepatic arterial problems (7%), hepatic vein stenosis (5%), portal vein stenosis (13%), biliary stricture (18%), intestinal perforation (3%). The overall rejection rate was 31%. Cytomegalovirus infection and Epstein-Barr virus disease were observed in 26% and 5%, respectively. Retransplantation was performed five times in four patients; the main cause was hepatic vein stenosis (n = 3). Four patients died; the main cause was gastrointestinal perforation (n = 2). The body height of Alagille syndrome patients less than 2 years old significantly improved compared with older patients after LDLT. The 1-, 5-, and 10-year patient survival rates were 98%, 97%, and 97%, respectively. Conclusions: LDLT for CLD is an effective treatment with excellent long-term outcomes. [Copyright &y& Elsevier]

Abstract: Background: Excessive portal pressure at an early stage after living-donor liver transplantation (LDLT) can damage sinusoidal endothelial cells and hepatocytes through shear stress leading to graft failure, or hepatic arterial complications due to low hepatic artery flow from a hepatic arterial buffer response. We encountered a case in which excessive portal vein flow was observed from an early stage after pediatric LDLT. The hepatic artery flow decreased due to a hepatic arterial buffer response. Case report: A 6-month-old boy with biliary atresia showed excessive portal vein flow early after LDLT with a decreasing hepatic artery flow without anastomotic stenosis from postoperative day 3. The PV flow gradually exhibited a decrease at approximately postoperative day 8 and, similtaneously, hepatic artery flow exhibited improvement. Conclusion: Because excessive portal pressure after LDLT is reversible, it has been suggested that it may be possible to prevent the progress of hepatic arterial complications if temporary portal pressure modulation can be performed for cases among the high-risk group for hepatic arterial complications. [Copyright &y& Elsevier]

Abstract: Background: There have been few reports on the management of intra-abdominal drains after living donor liver transplantation (LDLT). We retrospectively investigated changes in ascitic data related to management of an intra-abdominal drain. Patients and methods: Between March 2008 and June 2009, we performed 28 LDLT. On the first and the fifth postoperative day (POD) after LDLT , we examined the number of ascites cells and cell fractions as well as performed biochemical examination and cultures. Results: The day of removal of the drain for massive ascites (10 mL/kg/d or more) was 14.2 ± 5.4 POD; for less than 10 mL/kg/d it was 8.7 ± 1.9 POD (P < .001). Nine patients were ascites culture positive; long-term placement of the drain caused an infection in two patients. Conclusions: When the amount of ascitic fluid on the fifth POD after LDLT was small, it was important to assess the properties of the ascitic fluid because of the possibility of a drain infection or of poor drainage. If the ascitic neutrophil count is less than 250/mm3 or the examined ascites is normal, intra-abdominal drains should be removed. [Copyright &y& Elsevier]

Abstract: Objectives: To describe our experience with 126 consecutive living-donor liver transplantation (LDLT) procedures performed because of biliary atresia and to evaluate the optimal timing of the operation. Patients and Methods: Between May 2001 and January 2010,126 patients with biliary atresia underwent 130 LDLT procedures. Mean (SD) patient age was 3.3 (4.2) years, and body weight was 13.8 (10.7) kg. Donors included 64 fathers, 63 mothers, and 3 other individuals. The left lateral segment was the most commonly used graft (75%). Patients were divided into 3 groups according to body weight: group 1, less than 8 kg (n = 40); group 2,8 to 20 kg (n = 63); and group 3, more than 20 kg (n = 23). Medical records were reviewed retrospectively. Follow up was 4.5 (2.7) years. Results: All group 3 donors underwent left lobectomy, and all group 1 donors underwent left lateral segmentectomy. No donors required a second operation or died. Comparison of the 3 groups demonstrated that recipient Pediatric End-Stage Liver Disease score in group 1 was highest, operative blood loss in group 2 was lowest (78 mL/kg), and operative time in group 3 was longest (1201 minutes). Hepatic artery complications occurred more frequently in group 1 (17.9%), and biliary stenosis (43.5%) and gastrointestinal perforation (8.7%) occurred more frequently in group 3. The overall patient survival rates at 1, 5, and 9 years was 98%, 97%, and 97%, respectively. Five-year patient survival rate in groups 1,2, and 3 were 92.5%, 100%, and 95.7%, respectively. Gastrointestinal perforation (n = 2) was the primary cause of death. Conclusions: Living-donor liver transplantation is an effective treatment of biliary atresia, with good long-term outcome. It seems that the most suitable time to perform LDLT to treat biliary atresia is when the patient weighs 8 to 20 kg. [Copyright &y& Elsevier]

Although waterfowl are less susceptible to Newcastle disease (ND) virus (NDV) infection compared with chickens and turkeys, lethal ND in waterfowl has been sporadically reported. Factors underlying the high pathogenicity of certain NDV strains in waterfowl remain unclear. In ducks, the NDV 9a5b isolate shows low pathogenicity while the d5a20b isolate shows high pathogenicity. This study aimed to identify the definitive lesions that led to the lethal virulence of d5a20b by comparing the histopathology of 9a5b- or d5a20b-inoculated ducks in order to elucidate lesions related to the enhanced pathogenicity of certain NDV strains in ducks. Herein, 7-day-old ducks were intranasally inoculated with either 9a5b or d5a20b NDV strains. The neurological signs were more severe in the d5a20b-inoculated group than in the 9a5b-inoculated group. Ducks in the d5a20b-inoculated group exhibited more severe lymphoid depletion in immune organs than those in the 9a5b-inoculated group, which may have caused an immunosuppressive state in the d5a20b-inoculated ducks. Ducks in the d5a20b-inoculated group had more severe nonsuppurative encephalitis with increased NDV nucleoprotein than those in the 9a5b-inoculated group. Additionally, pancreatic necrosis, with intralesional NDV nucleoprotein, was more severe in the d5a20b-inoculated group than in the 9a5b-inoculated group. Our results showed that the immune organs, brain, and pancreas were significant targets of the NDV d5a20b infection in ducks., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Situs inversus complicates diagnosis and treatment due to the mirrored organ placement in relation to normal anatomy. This report describes a 78-year-old female patient with situs inversus totalis who underwent laparoscopic cholecystectomy and laparoscopic common bile duct exploration for cholecystolithiasis and choledocholithiasis. Utilizing the "French mirror technique" for port placement, the surgeon adeptly mirrored standard maneuvers with a 2-mm needle forceps in the left hand and a 5-mm forceps in the right in a reversed anatomical setting. This technique maintained familiar hand movements, despite the patient's unique anatomy. The surgeon applied transcystic ductal bile duct exploration, using choledochoscopy for duct exploration and a basket catheter for stone removal. Laparoscopic cholecystectomy and common bile duct exploration through the transcystic ductal route are viable and effective for patients with situs inversus., (© 2024 Asia Endosurgery Task Force and Japan Society of Endoscopic Surgery and John Wiley & Sons Australia, Ltd.)

A newborn male Holstein calf developed a nodular enlargement at the tip of the tongue. Histopathological examination of the mass revealed predominant proliferating small, round, spindloid or polygonal neoplastic cells with scattered myoblast- and myotube-like cells and multinuclear giant cells. Phosphotungstic acid haematoxylin staining revealed cytoplasmic cross-striations in a few neoplastic cells. Neoplastic cells were immunopositive for vimentin, desmin, myoD1, myogenin, myoglobin and α-smooth muscle actin. The mass was diagnosed as embryonal rhabdomyosarcoma. To the best of our knowledge, this is the first reported case of bovine congenital lingual rhabdomyosarcoma, which is rare in animals., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Gene therapy using adeno-associated virus (AAV)-based vectors has become a realistic therapeutic option for hemophilia. We examined the potential of a novel engineered liver-tropic AAV3B-based vector, AAV.GT5, for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, another bioengineered vector used in a clinical trial. However, liver transduction following intravenous injection of these vectors was similar in mice with a humanized liver and in macaques. This discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the capsid. Bypassing systemic clearance with the intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced liver transduction in pigs and macaques. AAV.GT5 did not develop neutralizing antibodies (NAbs) in two of four animals, while AAV-Spark100 induced serotype-specific NAbs in all macaques tested (4 of 4). The NAbs produced after AAV-Spark100 administration were relatively serotype specific, and challenge with AAV.GT5 through the hepatic artery successfully boosted liver transduction in one animal previously administered AAV-Spark100. In summary, AAV.GT5 showed different vector kinetics and NAb induction compared with AAV-Spark100, and intra-hepatic vascular administration may minimize the vector dose required and vector dissemination., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Resuscitative endovascular balloon occlusion of the aorta (REBOA) increases proximal blood pressure while inducing distal ischemia of visceral organs. The evaluation of distal ischemia severity during REBOA is a prerequisite for safe resuscitation of haemorrhagic shock patients with REBOA. We evaluated changes in blood flow and organ perfusion due to the degree of occlusion using dynamic 4D-computed tomography (CT). We compared the results with those of a previous study on euvolemic status. Delayed enhancement of the inferior vena cava (IVC) without retrograde flow was observed in the 4D-volume rendering images in the high-degree occlusion. The time-density curve (TDC) of the liver parenchyma (liver perfusion) and superior mesenteric vein (SMV) demonstrated a decreased peak density and a delayed peak in high-degree occlusion. The change rate of the area under the TDC of the liver and SMV decreased linearly as the degree of occlusion increased (PV, Y = -1.071*X + 106.8, r 2  = 0.972, P = 0.0003; liver, Y = -1.050*X + 101.8, r 2  = 0.933, P = 0.0017; SMV, Y = -0.985*X + 100.3, r 2  = 0.952, P = 0.0009). Dynamic 4D-CT revealed less severe IVC congestion during P-REBOA in haemorrhagic shock than in euvolemia. Analyses of TDC of the liver and SMV revealed a linear change in organ perfusion, regardless of intravascular volume., (© 2022. The Author(s).)

Objective : To examine early histologic changes in the aorta exposed to bicuspid flow. Material and Methods : A porcine bicuspid aortopathy model was developed by suturing aortic cusps. Of nine pigs, eight underwent sham surgery (n=3) or bicuspidalization (n=5); one was used as an intact control. Wall shear stress (WSS) was assessed by computational fluid dynamics (CFD). Animals were exposed to normal or bicuspid flow for 48 h and were then euthanized for histologic examinations. Results : No animal died intraoperatively. One animal subjected to bicuspidalization died of respiratory failure during postoperative imaging studies. Echocardiography showed the aortic valve area decreased from 2.52±1.15 to 1.21±0.48 cm 2 after bicuspidalization, CFD revealed increased maximum WSS (10.0±5.2 vs. 54.0±25.7 Pa; P=0.036) and percentage area of increased WSS (>5 Pa) in the ascending aorta (30.3%±24.1% vs. 81.3%±13.4%; P=0.015) after bicuspidalization. Hematoxylin-eosin staining and transmission electron microscopy showed subintimal edema and detached or degenerated endothelial cells following both sham surgery and bicuspidalization, regardless of WSS distribution. Conclusion : A bicuspid aortic valve appears to increase aortic WSS. The endothelial damage observed might have been related to non-pulsatile flow (cardiopulmonary bypass). Chronic experiments are needed to clarify the relationship between hemodynamic stress and development of bicuspid aortopathy., Competing Interests: Disclosure StatementThe authors have no conflicts of interest to disclose., (© 2022 The Editorial Committee of Annals of Vascular Diseases.)

Purpose: The aim of this study was to evaluate both the intestinal mucosa staple line integrity and anastomotic leak pressure after healing in a porcine survival model., Methods: We used two suture models using two different size staples (incomplete mucosal closure model: group G [staple height 0.75 mm], complete mucosal closure model: group B [staple height 1.5 mm]) in the porcine ileum. Five staple lines were created in each group made in the ileum for each model, and the staple sites harvested on days 0, 2, and 7. The leak pressure at the staple site was measured at each time point., Results: On day 0, the leak pressure for group G (79.5 mmHg) was significantly lower than that for group B (182.3 mmHg) (p < 0.01). On days 2 and 7, there was no significant difference between groups G and B (171 mmHg and 175.5 mmHg on day 2, 175.5 mmHg and 175.5 mmHg on day 7, p > 0.05). The histological findings in both groups showed similar healing at postoperative days 2 and 7., Conclusion: The integrity of the mucosal staple lines was associated with the postoperative leak pressure on day 0. However, there was no association with the leak pressure at two days or more postoperatively in a porcine model., (© 2021. Springer Nature Singapore Pte Ltd.)

Introduction: Resuscitative endovascular balloon occlusion of the aorta (REBOA) increases proximal arterial pressure, but may also induce life-threatening distal ischemia. Partial REBOA (P-REBOA) is thought to mitigate distal ischemia during aortic occlusion. However, feasible indicators of the degree of P-REBOA remain inconsistent. We hypothesised percent balloon volume could be a substitute for pressure measurements of gradients during P- REBOA. This study aimed to compare balloon volume and arterial pressure gradient, and analysed with intra-balloon pressure and balloon shape., Methods: Proximal (carotid) and distal (femoral) arterial pressures were recorded and a 7-Fr REBOA catheter was placed in four swine. Total REBOA was defined as a cessation of distal pulse pressure and maximum balloon volume was documented. The balloon volume was titrated by 20% increments of maximum capacity to adjust the degree of P-REBOA. The distal/proximal arterial pressure gradient and the intra-balloon pressures were also recorded. The changes in shape and the cross-sectional area of the balloon were evaluated with computed tomography (CT) images., Results: The proximal mean arterial pressure (MAP) plateaued after 60% balloon volume; meanwhile, distal pulse pressure was still left. The balloon pressure was traced with proximal MAP before contact with aortic wall. The balloon shape changed unevenly from "cone" to "spindle" shape, although the balloon cross-sectional area of the mid-segment linearly increased., Conclusion: Monitoring distal pressure and titrating percent balloon volume is feasible to manage P-REBOA. In this experiment, 60% balloon volume was enough inflation to elevate central pressure allowing distal perfusion. The intra-balloon pressure was not reliable due to the strong influence of proximal MAP and uneven change of the balloon shape., (© 2019. Springer-Verlag GmbH Germany, part of Springer Nature.)

Glucose transporter 1 deficiency syndrome (GLUT1DS) is caused by haplo-insufficiency of SLC2A1, which encodes GLUT1, resulting in impaired hexose transport into the brain. Previously, we generated a tyrosine-mutant AAV9/3 vector in which SLC2A1 was expressed under the control of the endogenous GLUT1 promoter (AAV-GLUT1), and confirmed the improved motor function and cerebrospinal fluid glucose levels of Glut1-deficient mice after cerebroventricular injection of AAV-GLUT1. In preparation for clinical application, we examined the expression of transgenes after intra-cisterna magna injection of AAV-GFP (tyrosine-mutant AAV9/3-GFP with the CMV promoter) and AAV-GLUT1. We injected AAV-GFP or AAV-GLUT1 (1.63 × 10 12 vector genomes/kg) into the cisterna magna of pigs to compare differential promoter activity. After AAV-GFP injection, exogenous GFP was expressed in broad areas of the brain and peripheral organs. After AAV-GLUT1 injection, exogenous GLUT1 was expressed predominantly in the brain. At the cellular level, exogenous GLUT1 was mainly expressed in the endothelium, followed by glia and neurons, which was contrasted with the neuronal-predominant expression of GFP by the CMV promotor. We consider intra-cisterna magna injection of AAV-GLUT1 to be a feasible approach for gene therapy of GLUT1DS.

The severe shortage of donor hearts hampered the cardiac transplantation to patients with advanced heart failure. Therefore, cardiac regenerative therapies are eagerly awaited as a substitution. Human induced pluripotent stem cells (hiPSCs) are realistic cell source for regenerative cardiomyocytes. The hiPSC-derived cardiomyocytes are highly expected to help the recovery of heart. Avoidance of teratoma formation and large-scale culture of cardiomyocytes are definitely necessary for clinical setting. The combination of pure cardiac spheroids and gelatin hydrogel succeeded to recover reduced ejection fraction. The feasible transplantation strategy including transplantation device for regenerative cardiomyocytes are established in this study., Competing Interests: This work was supported by the Highway Program for Realization of Regenerative Medicine (17bm054006h0007 [to Dr. Fukuda]) and the Research Project for Practical Applications of Regenerative Medicine (17bk010462h0001 [to Dr. Fukuda]) from the Japan Agency for Medical Research and Development, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (nos. 15K09098 [to Dr. Kanazawa], 16K09507 [to Dr. Fujita], 19H03660 [to Dr. Fujita], 17H05067 [to Dr. Tohyama], 18K15903 [to Dr. Nakajima]). Drs. Kanazawa, Tohyama, Fukuda, and Fujita have patents related to this work. Drs. Tohyama, Shimizu, Kanazawa, Fukuda, and Fujita own equity in Heartseed, Inc. Dr. Tohyama is an advisor of Heartseed, Inc. Dr. Fukuda is a co-founder and CEO of Heartseed, Inc.; and receives a salary from Heartseed, Inc. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2021 The Authors.)

Adeno-associated virus (AAV) vectors can transduce hepatocytes efficiently in vivo in various animal species, including humans. Few reports, however, have examined the utility of pigs in gene therapy. Pigs are potentially useful in preclinical studies because of their anatomical and physiological similarity to humans. Here, we evaluated the utility of microminipigs for liver-targeted gene therapy. These pigs were intravenously inoculated with an AAV8 vector encoding the luciferase gene, and gene expression was assessed by an in vivo imaging system. Robust transgene expression was observed almost exclusively in the liver, even though the pig showed a low-titer of neutralizing antibody (NAb) against the AAV8 capsid. We assessed the action of NAbs against AAV, which interfere with AAV vector-mediated gene transfer by intravascular delivery. When a standard dose of vector was administered intravenously, transgene expression was observed in both NAb-negative and low-titer (14×)-positive subjects, whereas gene expression was not observed in animals with higher titers (56×). These results are compatible with our previous observations using nonhuman primates, indicating that pigs are useful in gene therapy experiments, and that the role of low-titer NAb in intravenous administration of the AAV vector shows similarities across species.

Buckybowls bearing a C 70 fragment having two alkoxy groups were synthesized and their structural and optical properties were investigated by single crystal X-ray analysis and UV-vis spectroscopy. In the synthesis of dioxole derivative 5b , the regioisomer 5c was also produced. The yield of 5c was increased by increasing the reaction temperature, indicating that the rearrangement might involve the equilibrium between the Pd(IV) intermediates through C-H bond activation., (Copyright © 2020, Yakiyama et al.; licensee Beilstein-Institut.)

Resuscitative endovascular balloon occlusion of the aorta (REBOA) increases proximal pressure, and simultaneously induces distal ischemia. We aimed to evaluate organ ischemia during partial REBOA (P-REBOA) with computed tomography (CT) perfusion in a swine model. The maximum balloon volume was recorded as total REBOA when the distal pulse pressure ceased. The animals (n = 4) were scanned at each 20% of the maximum balloon volume, and time-density curve (TDC) were analysed at the aorta, portal vein (PV), liver parenchyma, and superior mesenteric vein (SMV, indicating mesenteric perfusion). The area under the TDC (AUTDC), the time to peak (TTP), and four-dimensional volume-rendering images (4D-VR) were evaluated. The TDC of the both upper and lower aorta showed an increased peak and delayed TTP. The TDC of the PV, liver, and SMV showed a decreased peak and delayed TTP. The dynamic 4D-CT analysis suggested that organ perfusion changes according to balloon volume. The AUTDC at the PV, liver, and SMV decreased linearly with balloon inflation percentage to the maximum volume. 4D-VR demonstrated the delay of the washout in the aorta and retrograde flow at the inferior vena cava in the highly occluded status.

Background: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a viable resuscitation approach for a subdiaphragmatic injury that can regulate arterial blood flow. On the other hand, the evaluation of venous or portal venous blood flow during REBOA remains insufficient because invasive cannulation or exposure of the vessel may affect the blood flow, and Doppler echography is highly operator-dependent. However, phase contrast magnetic resonance imaging has enabled accurate evaluation and noninvasive measurement. This study aimed to investigate the change of venous and portal venous blood flow during REBOA in a porcine model., Methods: Seven pigs were anesthetized, and a REBOA catheter was placed. The blood flows of the inferior vena cava (IVC), hepatic vein (HV), portal vein (PV), and superior vena cava (SVC) were measured using phase contrast magnetic resonance imaging, in both the balloon deflated (no-REBOA) and fully balloon inflated (REBOA) states. Mean arterial pressure (MAP), central venous pressure, cardiac index, and systemic vascular resistance index were measured., Results: The blood flows of the suprahepatic, infrahepatic, and distal IVC, HV, and PV in the no-REBOA state were 1.40 ± 0.36 L·min, 0.94 ± 0.16 L·min, 0.50 ± 0.19 L·min, 0.060 ± 0.018 L·min, and 0.32 ± 0.091 L·min, respectively. The blood flow of each section in the REBOA condition was significantly decreased at 0.41 ± 0.078 (33% of baseline), 0.15 ± 0.13 (15%), 0.043 ± 0.034 (9%), 0.029 ± 0.017 (37%), and 0.070 ± 0.034 L·min (21%), respectively. The blood flow of the SVC increased significantly in the REBOA condition (1.4 ± 0.63 L·min vs. 0.53 ± 0.14 L·min [257%]). Mean arterial pressure, central venous pressure, cardiac index, and systemic vascular resistance index were significantly increased after REBOA inflation., Conclusion: Resuscitative endovascular balloon occlusion of the aorta decreased blood flows of the IVC, HV, and PV and increased blood flow of the SVC. This result could be explained by the collateral flow from the lower body to the SVC. A better understanding of the effect of REBOA on the venous and portal venous systems may help control liver injury.

Background: Induced pluripotent stem cell (iPSC)‒based regenerative therapy is a promising strategy for cardiovascular disease treatment; however, the method is limited by the myocardial retention of grafted iPSCs. Thus, an injection protocol that efficiently introduces and retains human iPSC-derived cardiomyocytes (hiPSC-CMs) within the myocardium is urgently needed. The objective of the present study was to develop a method to improve the retention of hiPSCs in the myocardium for cardiac therapy., Methods: We efficiently produced hiPSC-CM spheroids in 3-dimensional (3D) culture using microwell plates, and developed an injection device for optimal 3D distribution of the spheroids in the myocardial layer. Device biocompatibility was assessed with purified hiPSC-CM spheroids. Device effectiveness was evaluated in 10- to 15-month-old farm pigs (n = 15) and 5- to 24-month-old micro-minipigs (n = 20). The pigs were euthanized after injection, and tissues were harvested for retention and histologic analysis., Results: We demonstrated an injection device for direct intramyocardial transplantation of hiPSC-CM spheroids from large-scale culture. The device had no detrimental effects on cell viability, spheroid shape, or size. Direct epicardial injection of spheroids mixed with gelatin hydrogel into beating porcine hearts using this device resulted in better distribution and retention of transplanted spheroids in a layer within the myocardium than did conventional needle injection procedures., Conclusions: The combination of the newly developed transplant device and spheroid formation promotes the retention of transplanted CMs. These findings support the clinical application of hiPSC-CM spheroid‒based cardiac regenerative therapy in patients with heart failure., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20-100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=-0.97, P<0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG +/- pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.

Purpose: When living donor liver transplantation (LDLT) is performed on small infant patients, the incidence of hepatic artery complications (HACs) is high. Here, we present a retrospective analysis that focuses on our surgical procedure for hepatic arterial reconstruction and the outcomes of monosegmental LDLT., Methods: Of the 275 patients who underwent LDLT between May 2001 and December 2015, 13 patients (4.7 %) underwent monosegmental LDLT. Hepatic artery reconstruction was performed under a microscope. The size discrepancy between the graft and the recipient's abdominal cavity was defined as the graft to recipient distance ratio (GRDR) between the left hepatic vein and the portal vein (PV) bifurcation on a preoperative computed tomography scan. HACs were defined as hepatic arterial hypoperfusion., Results: Recipient hepatic arteries were selected for the branch patch technique in five cases (38.5 %), and the diameter was 2.2 ± 0.6 mm. The anastomotic approaches selected were the dorsal position of the PV in seven cases (53.8 %) and the ventral position in six, and the GRDRs were 2.8 ± 0.4 and 1.9 ± 0.5, respectively (p = 0.012). The incidence rate of HACs caused by external factors, such as compression or inflammation around the anastomotic site, was significantly higher in monosegmental than in non-monosegmental graft recipients (15.4 vs. 1.1 %, p < 0.001)., Conclusion: Although monosegmental graft recipients experienced HACs caused by external factors around the anastomotic field, hepatic arterial reconstruction could be safely performed. Important components of successful hepatic arterial reconstructions include the employment of the branch patch technique and the selection of the dorsal approach.

Animal models of thrombocytopenia are indispensable for evaluating the in vivo efficacy of hemostatic agents, cryopreserved platelets, and artificial platelets, but no large animal models are available. In this study, we generated a swine model of acute thrombocytopenia with prolonged bleeding times by administering the chemotherapeutic drug busulfan. First, we tested multiple doses of busulfan (4, 6, and 8 mg/kg) in pigs, and found that 6 mg/kg of busulfan is an optimal dose for producing a safe and moderate thrombocytopenia, with a platelet count of less than 30,000/µl. The pigs administered 6 mg/kg of busulfan (n=8) reached half their initial counts at day 7, counts below 30,000/µl at day 12, and their nadirs at day 15 (on average). The minimal platelet count was 14,000/µl. With this dose of busulfan (6 mg/kg), bleeding times were significantly prolonged in addition to the decrease in platelet counts (r=-0.63, P<0.01), while there were no cases of apparent hemorrhage. White blood cell counts were maintained at over 5,000/µl, and there were no infections or other adverse events including anemia or appetite or body weight loss. All pigs were sacrificed on day 16, with subsequent examination showing a significant reduction in cellularity and colony-forming units in the bone marrow, indicating that thrombocytopenia was the result of myelosuppression. In summary, administration with 6 mg/kg of busulfan induces safe and moderate thrombocytopenia with a prolonged bleeding time in swine.

Background: Live tissue models are considered the most useful simulation for training in the management for hemostasis of penetrating injuries. However, these models are expensive, with limited opportunities for repetitive training. Ex-vivo models using tissue and a fluid pump are less expensive, allow repetitive training and respect ethical principles in animal research. The purpose of this study is to objectively evaluate the effectiveness of ex-vivo training with a pump, compared to live animal model training. Staff surgeons and residents were divided into live tissue training and ex-vivo training groups. Training in the management of a penetrating cardiac injury was conducted for each group, separately. One week later, all participants were formally evaluated in the management of a penetrating cardiac injury in a live animal., Results: There are no differences between the two groups regarding average years of experience or previous trauma surgery experience. All participants achieved hemostasis, with no difference between the two groups in the Global Rating Scale score (ex-vivo: 25.2 ± 6.3, live: 24.7 ± 6.3, p = 0.646), blood loss (1.6 ± 0.7, 2.0 ± 0.6, p = 0.051), checklist score (3.7 ± 0.6, 3.6 ± 0.9, p = 0.189), or time required for repair (101 s ± 31, 107 s ± 15, p = 0.163), except overall evaluation (3.8 ± 0.9, 3.4 ± 0.9, p = 0.037). The internal consistency reliability and inter-rater reliability in the Global Rating Scale were excellent (0.966 and 0.953 / 0.719 and 0.784, respectively), and for the checklist were moderate (0.570 and 0.636 / 0.651 and 0.607, respectively). The validity is rated good for both the Global Rating Scale (Residents: 21.7 ± 5.6, Staff: 28.9 ± 4.7, p = 0.000) and checklist (Residents: 3.4 ± 0.9, Staff Surgeons: 3.9 ± 0.3, p = 0.003). The results of self-assessment questionnaires were similarly high (4.2-4.9) with scores in self-efficacy increased after training (pre: 1.7 ± 0.8, post: 3.2 ± 1.0, p = 0.000 in ex-vivo, pre: 1.9 ± 1.0, post: 3.7 ± 0.7, p = 0.000 in live). Scores comparing pre-training and post-evaluation (pre: 1.7 ± 0.8, post: 3.7 ± 0.9, p = 0.000 in ex-vivo, pre: 1.9 ± 1.0, post: 3.8 ± 0.7, p = 0.000 in live) were increased., Conclusion: Training with an ex-vivo model and live tissue training are similar for the management of a penetrating cardiac injury, with increased self-efficacy of participants in both groups. The ex-vivo model is useful to learn hemostatic skills in trauma surgery.

Background: Hepatic artery complications (HAC) are a serious complication in pediatric liver transplant recipients because its incidence is high and it can occasionally lead to graft liver failure. We herein present a retrospective analysis of our 10-year experience with pediatric living donor liver transplantation (LDLT) focusing on the risk factors and treatments for HAC., Methods: Between May 2001 and November 2011, 209 LDLTs were performed for 203 pediatric recipients. We performed the multivariate analyses to identify the factors associated with HAC and showed the therapeutic strategy and outcome for HAC., Results: The overall incidence of HAC was 7.2%, and the graft survival of recipients with HAC was 73.3%. The multivariate analysis showed that the pediatric end-stage liver disease score (≥20), post-transplant laparotomy except for HAC treatment and extra-anatomical hepatic artery reconstruction were independent risk factors for HAC (P = 0.020, P = 0.015 and P = 0.002, respectively). Eleven surgical interventions and 13 endovascular interventions were performed for 15 recipients with HAC. The serum aspartate aminotransferase levels pre- and post-treatment for HAC were significantly higher in the surgical group than in the endovascular group (P = 0.016 and P = 0.022, respectively)., Conclusions: It is important for recipients with risk factors to maintain strict post-transplant management to help prevent HAC and detect it in earlier stages. Endovascular intervention can be a less invasive method for treating HAC than surgical intervention, and can be performed as an early treatment., (© 2013 Japanese Society of Hepato-Biliary-Pancreatic Surgery.)

Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.

Background: The goal of this study is to examine changes in pancreatic perfusion due to pneumoperitoneum using perfusion CT in vivo., Methods: Three pigs were studied. Under general anesthesia, pneumoperitoneum was induced to 16 mm Hg. Perfusion CT scans were acquired at a rate of 1 image per 2 seconds for 60 seconds. Scans were repeated 5 days later without pneumoperitoneum using the same protocol, in the same animals. The time density curve, color map, peak enhancement, time to peak, blood flow, blood volume, and permeability were evaluated., Results: In the presence of pneumoperitoneum, peak enhancement in radiodensity was decreased and time to peak was increased, and both blood flow and blood volume decreased. However, there was no consistent change in permeability observed., Conclusion: This study demonstrates that pneumoperitoneum quantitatively results in decreased blood flow and blood volume to the pancreas in an in vivo animal model.

In developing therapeutic alternatives to liver transplantation, we have used the strategy of applying a small intestinal segment as a scaffold for hepatocyte transplantation and also as a portocaval shunt (PCS) system to address both liver dysfunction and portal hypertension. The aim of this study was to investigate the feasibility of such an intestinal segment in animal models. Hepatocytes isolated from luciferase-transgenic Lewis rats were transplanted into jejunal segments of wild-type Lewis rats with mucosa removal without PCS application. Luciferase-derived luminescence from transplanted hepatocytes was stably detected for 30 days. Then, we performed autologous hepatocyte transplantation into the submucosal layer of an isolated and vascularized small intestinal segment in pigs. Transplanted hepatocytes were isolated from the resected left-lateral lobe of the liver. On day 7, hepatocyte clusters and bile duct-like structures were observed histologically. To create an intestinal PCS system in pigs, an auto-graft of the segmental ileum and interposing vessel graft were anastomosed to the portal vein trunk and inferior vena cava. However, thrombi were observed in vessels of the intestinal PCSs. We measured the correlation between infusion pressure and flow volume in whole intestines ex vivo in both species and found that the high pressure corresponding to portal hypertension was still insufficient to maintain the patency of the intestinal grafts. In conclusion, we demonstrated the feasibility of the small intestine as a scaffold for hepatocyte transplantation in rat and pig models, but PCS using an intestinal graft failed to maintain patency in a pig model.

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 ± 2.10-10.1 ± 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 ± 2.59-12.68 ± 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 ± 1.06-9.0 ± 2.37%) in the presence of NAbs (14-56× dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.

To improve the welfare of experimental animals, investigators seek to respect the 3R principle (Replacement, Reduction, and Refinement). Even when large animal studies are essential before moving to clinical trials, it is important to look for ways to reduce the number of experimental animals used. At the Center for the Development of Advanced Medical Technology, we consider 'medical' pigs to be ideal preclinical model systems.We have been using both wild-type and genetically modified pigs. We began using this approach about 10 years ago with a 'total pig system' to model human health and disease for the purposes of both medical skill education and the development of new devices and therapeutic strategies.At our Center, medical students and residents use pigs to gain experience with surgical skills and train for emergency procedures after appropriate simulation training. Senior clinicians have also used these models to advance the development of innovative tools for endo- and laparoscopic procedures. The Center focuses on translational research for organ transplantation and stem cell therapy. Several pig models have been established for liver, intestine, kidney, pancreas, and lung transplantation. Mesenchymal stromal cells have been established in green fluorescent protein- and red fluorescent protein-transgenic pigs and tested to trans-differentiate organogenesis. A program to establish induced pluripotent stem cells in the pig is ongoing at our Center.Here, we review our 10 years of activity in this field. Based on our experience in surgical education and research, experimental pigs are valuable models in translational research.

Background: The use of patients as the primary teaching modality for learning procedures is being questioned. While there have been advancements in the technology used for performing needle biopsies in both native and transplanted kidneys, there has been little advancement in teaching and training tools. We have developed a portable ex-vivo kidney, the Bento Kidney, using cryo-preserved porcine kidneys for teaching this procedure., Methods: The kidney is thawed, perfused by a pump, covered with skin for realistic haptic feedback, and then used with existing biopsy technology to teach the technique., Results: Thirty porcine kidneys were used in this pilot research, and nine were shipped to physicians at a distant facility. Renal biopsy was then performed using a core biopsy needle and ultrasound guidance. There was some leakage of fluid from all kidneys noted. All trainees felt that the model was realistic, and judged at a mean score of 8.7 (SD 0.8) on a scale of 1 (not useful) to 10 (very useful)., Conclusions: This feasibility study demonstrates that cryo-preserved porcine kidneys can be successfully used to teach and train renal biopsy techniques, and provides haptic feedback as well as realistic real-time ultrasound images. Further large scale studies are needed to demonstrate value from the educational point of view for nephrology and transplantation.

Background: Liver transplantation for biliary atresia is indicated whenever a Kasai portoenterostomy is considered unfeasible. However, the timing of liver transplantation in biliary atresia has not been precisely defined. Excessive shortening of hepatocellular telomeres may occur in patients with biliary atresia, and therefore, telomere length could be a predictor of hepatocellular reserve capacity., Methods: Hepatic tissues were obtained from 20 patients with biliary atresia who underwent LT and 10 age-matched autopsied individuals (mean age, 1.7 and 1.2 years, respectively). Telomere lengths were measured by Southern blotting and quantitative fluorescence in situ hybridization using the normalized telomere-centromere ratio. The correlation between the normalized telomere-centromere ratio for the hepatocytes in biliary atresia and the pediatric end-stage liver disease score was analyzed., Results: The median terminal restriction fragment length of the hepatic tissues in biliary atresia was not significantly different from that of the control (p = 0.425), whereas the median normalized telomere-centromere ratio of hepatocytes in biliary atresia was significantly smaller than that of the control (p < 0.001). Regression analysis demonstrated a negative correlation of the normalized telomere-centromere ratio with the pediatric end-stage liver disease score in biliary atresia (p < 0.001)., Conclusions: Telomere length analysis using quantitative fluorescence in situ hybridization could be an objective indicator of hepatocellular reserve capacity in patients with biliary atresia, and excessive telomere shortening supports the early implementation of liver transplantation.