Novel approach for reconstruction of the three-dimensional biliary system in decellularized liver scaffold using hepatocyte progenitors.

Reconstruction of the biliary system is indispensable for the regeneration of transplantable liver grafts. Here, we report the establishment of the first continuous three-dimensional biliary system scaffold for bile acid excretion using a novel method. We confirmed the preservation of the liver-derived extracellular matrix distribution in the scaffold. In addition, hepatocyte progenitors decellularized via the bile duct by slow-speed perfusion differentiated into hepatocyte- and cholangiocyte-like cells, mimicking hepatic cords and bile ducts, respectively. Furthermore, qRT-PCR demonstrated increased ALB, BSEP, and AQP8 expression, revealing bile canaliculi- and bile duct-specific genetic patterns. Therefore, we concluded that locally preserved extracellular matrices in the scaffold stimulated hepatic progenitors and provided efficient differentiation, as well as regeneration of a three-dimensional continuous biliary system from hepatic cords through bile ducts. These findings suggest that organ-derived scaffolds can be utilized for the efficient reconstruction of functional biliary systems.

Surgical Punctal Occlusion; Combined Lacrimal Canaliculi Cauterization and Punctal Suturing for Severe Dry Eye.

Purpose: To evaluate the treatment outcome of surgical punctal occlusion with combined canaliculi ablation and punctal suturing in patients with severe dry eye. Methods: Eleven eyes of seven patients were diagnosed with severe dry eye with decreased lacrimal secretion and were refractory to treatment with various eye drops and/or had repeatedly experienced loss of punctal plugs, and continued to experience subjective symptoms received surgical punctal occlusion. In 20 puncta, lacrimal canaliculi ablation was performed along the entire length of the lacrimal canaliculus where a diathermy needle could be inserted. After resection of the annulus fibrosus in the peri-punctal area, tight cross-stitch suturing of the puncta was performed with 8-0 absorbent thread. Visual acuity, corneal staining score according to the area (A) and density (D) classification, and Schirmer tear test (STT); tear break up time (tBUT); and subjective symptoms assessed by the University of North Carolina (UNC) and Dry Eye Management Scale were compared before and one year after surgery. Results: Recanalization occurred in 1/20 puncta (5.0% at month 5) in 1/11 eyes. Student's t-test showed significant improvement at one year compared with preoperative values for LogMAR value (P = 0.019), corneal staining score A (P = 0.00003) and D (P = 0.0003), STT (P = 0.004), and subjective symptoms (P = 0.015). No change was shown in tBUT and no serious adverse event occurred. Conclusion: This improved, minimally invasive surgical procedure has a low recanalization rate and achieves both objective and subjective improvements at one year.

Kynurenine promotes Calcitonin secretion and reduces cortisol in the Japanese flounder Paralichthys olivaceus.

Deep ocean water (DOW) exerts positive effects on the growth of marine organisms, suggesting the presence of unknown component(s) that facilitate their aquaculture. We observed that DOW suppressed plasma cortisol (i.e., a stress marker) concentration in Japanese flounder (Paralichthys olivaceus) reared under high-density condition. RNA-sequencing analysis of flounder brains showed that when compared to surface seawater (SSW)-reared fish, DOW-reared fish had lower expression of hypothalamic (i.e., corticotropin-releasing hormone) and pituitary (i.e., proopiomelanocortin, including adrenocorticotropic hormone) hormone-encoding genes. Moreover, DOW-mediated regulation of gene expression was linked to decreased blood cortisol concentration in DOW-reared fish. Our results indicate that DOW activated osteoblasts in fish scales and facilitated the production of Calcitonin, a hypocalcemic hormone that acts as an analgesic. We then provide evidence that the Calcitonin produced is involved in the regulatory network of genes controlling cortisol secretion. In addition, the indole component kynurenine was identified as the component responsible for osteoblast activation in DOW. Furthermore, kynurenine increased plasma Calcitonin concentrations in flounders reared under high-density condition, while it decreased plasma cortisol concentration. Taken together, we propose that kynurenine in DOW exerts a cortisol-reducing effect in flounders by facilitating Calcitonin production by osteoblasts in the scales.

A retrospective analysis for criteria of surgical intervention in Japanese Black calves with respiratory signs caused by perinatal rib fracture.

Medical records of 16 Japanese Black calves with respiratory signs associated with perinatal rib fracture were analyzed, retrospectively, to speculate criteria of surgical intervention for the disease. For this analysis, the severity of respiratory condition was classified into three grades: Grade 1, with no wheezing, Grade 2, with wheezing after excitement but not at rest and Grade 3, with wheezing at rest. Grade 1 (n=3) received only conservative management. Seven of Grade 2 (n=8) and all of Grade 3 (n=5) had surgical management. Clinical outcome was good in all of Grade 1, and the surgical success rate was 83% in other groups. Our result suggests that surgical intervention should be considered for cases with more the severity of Grade 2.

[A case of rectovaginal metastasis 6 years after initial resection of extra-gastrointestinal stromal tumor of vulvar origin].

A 53-year-old woman who had undergone excision of KIT-positive extra-gastrointestinal stromal tumor (EGIST) of the vulva 6 years ago presented to our hospital due to a positive fecal occult blood test. Colonoscopy revealed a submucosal tumor in the rectum ventral side. In addition, computed tomography and magnetic resonance imaging revealed a tumor in the rectovaginal septum. For diagnostic and therapeutic purposes, the tumor was resected via the perineal approach. The resected specimen analysis revealed a KIT-positive gastrointestinal stromal tumor (GIST). Following immunopathological and genetic mutation identifications, GIST of the rectovaginal septum from vulva EGIST metastasis was diagnosed. It is important to consider primary GIST and metastatic GIST as differential diagnoses in the case of a rectal submucosal tumor detected by endoscopy.

Surgical Treatment of a Retropharyngeal Abscess in a Japanese Black Cow.

A 17-month-old Japanese Black cow presented with inappetence, wheezing, dysphagia, and drooling. Radiography and ultrasonography revealed an oval, dorsal, pharyngeal mass, with an internal horizontal line demarcating the radiolucent area from the radio-opaque area. Upper airway endoscopy revealed pus-like deposits in the dorsal nasal passage, hyperemia, and edema in the dorsal pharynx, leading to swelling and airway obstruction. Manual palpation, after sedation, revealed a thickened mass surface, which was difficult to rupture with manual pressure. After inserting a linear sonographic probe through the mouth to establish the vascularity surrounding the mass and to identify a relatively thin-walled area, a trocar was pierced into the mass under endoscopic guidance, and the opening was enlarged manually. The mass was filled with stale pus-like material, which was removed manually. The abscess cavity was washed using saline and povidone-iodine. Day 1 post-surgery, dysphagia and wheezing disappeared. Day 16 post-surgery, endoscopy showed significant improvement in the airway patency. One year postoperatively, the owner reported that the cow had an uneventful recovery. For deep abscesses, such as bovine pharyngeal abscesses, it is important to perform a preoperative transoral Doppler ultrasonography to assess the vascularity and thickness of the abscess wall for safe trocar insertion and abscess drainage.

Intra-abdominal mass containing controlled internal drug release (CIDR) devices detected by CT in a Japanese Black cow.

This report describes an intra-abdominal mass caused by progesterone-containing controlled internal drug release (CIDR) devices in the abdominal cavity of a 28-month-old Japanese Black heifer presenting with infertility. Rectal examination revealed a mass containing a cartilage-like object in the pelvic cavity. Computed tomography revealed a mass containing two T-shaped foreign bodies. Laparotomy was performed to remove the mass containing the foreign bodies. The heifer was healthy at discharge seven days post-surgery. This case adds CIDR device as a new etiology for intra-abdominal masses. Furthermore, it highlights the usefulness of CT in diagnosing deep abdominal lesions.

Decellularized Organ-Derived Scaffold Is a Promising Carrier for Human Induced Pluripotent Stem Cells-Derived Hepatocytes.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for elucidating disease pathology and therapy. The mass supply of hiPSC-derived cells is technically feasible. Carriers that can contain a large number of hiPSC-derived cells and evaluate their functions in vivo-like environments will become increasingly important for understanding disease pathogenesis or treating end-stage organ failure. hiPSC-derived hepatocyte-like cells (hiPSC-HLCs; 5 × 108) were seeded into decellularized organ-derived scaffolds under circumfusion culture. The scaffolds were implanted into immunodeficient microminiature pigs to examine their applicability in vivo. The seeded hiPSC-HLCs demonstrated increased albumin secretion and up-regulated cytochrome P450 activities compared with those in standard two-dimensional culture conditions. Moreover, they showed long-term survival accompanied by neovascularization in vivo. The decellularized organ-derived scaffold is a promising carrier for hiPSC-derived cells for ex vivo and in vivo use and is an essential platform for regenerative medicine and research.

Identification of domestic cat hepadnavirus from a cat blood sample in Japan.

The hepatitis B virus (Hepadnaviridae) induces chronic hepatitis and hepatic cancer in humans. A novel domestic cat hepadnavirus (DCH) was recently identified in several countries, however, the DCH infection status of cats in Japan is unknown. Therefore, we investigated the DCH infection rate of 139 cat samples collected in Japan. We identified one positive blood sample (0.78%) from a 17-year-old female cat with chronically elevated alanine aminotransferase. Phylogenetic analysis demonstrated that the DCH strain identified in this study is genetically different from strains in other countries. Further investigations are required to elucidate the evolution of DCH and the impact of DCH infection on hepatic diseases in domestic cats.

An organ-derived extracellular matrix triggers in situ kidney regeneration in a preclinical model.

It has not been considered that nephrons regenerate in adult mammals. We present that an organ-derived extracellular matrix in situ induces nephron regeneration in a preclinical model. A porcine kidney-derived extracellular matrix was sutured onto the surface of partial nephrectomy (PN)-treated kidney. Twenty-eight days after implantation, glomeruli, vessels, and renal tubules, characteristic of nephrons, were histologically observed within the matrix. No fibrillogenesis was observed in the matrix nor the matrix-sutured kidney, although this occurred in a PN kidney without the matrix, indicating the structures were newly induced by the matrix. The expression of renal progenitor markers, including Sall1, Six2, and WT-1, within the matrix supported the induction of nephron regeneration by the matrix. Furthermore, active blood flow was observed inside the matrix using computed tomography. The matrix provides structural and functional foundations for the development of cell-free scaffolds with a remarkably low risk of immune rejection and cancerization.

Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model.

Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.

[A case of upper gastrointestinal bleeding due to a ruptured gastric aneurysm caused by arteriosclerosis derived from primary aldosteronism-induced hypertension].

A 77-year-old woman with a history of hypertension underwent upper gastrointestinal endoscopy for hematemesis, and vasodilation with erosion was detected. Contrast-enhanced computed tomography and abdominal angiography revealed a gastric aneurysm penetrating the stomach wall. Endoscopic hemostasis was thought to be not applicable to a ruptured aneurysm. Transcatheter arterial embolization was not performed because of technical difficulties. Therefore, local gastric resection was performed. It was later determined that the cause of the persistent hypertension was primary aldosteronism, which was thought to cause arteriosclerotic obstruction of the celiac, common hepatic, and splenic arteries, resulting in the formation of the gastric arterial aneurysm. In the case of upper gastrointestinal bleeding, keeping primary aldosteronism in mind as a differential diagnosis is important to make a precise diagnosis and suitable treatment selection.

Decellularization of canine kidney for three-dimensional organ regeneration.

BACKGROUND AND AIM: Kidney regeneration is required for dogs with end-stage renal failure. Decellularization is one of the bioengineering techniques, which involves the removal of all tissue cells and cellular components and conservation of the extracellular matrix (ECM). Studies in rats have shown that decellularized kidney has regenerative potential; however, there are no reports on renal decellularization in dogs. Here, we showed the decellularization of the canine kidney. MATERIALS AND METHODS: The renal artery of the cadaveric canine kidney was cannulated and the whole kidney was frozen at -80°C. After completely thawing, it was perfused with physiological saline and sodium dodecyl sulfate (0.5%, 6 h) through the cannulated renal artery to achieve decellularization. To assess the efficiency of the decellularization protocol, histological and immunohistochemical analysis of decellularized kidney was performed. RESULTS: The results of hematoxylin and eosin (H and E) staining revealed that the decellularized canine kidney had no apparent cellular components. In addition, 4',6-diamidino-2-phenylindole (DAPI) staining showed no visible nuclear components within the whole decellularized kidney. Therefore, both H and E and DAPI staining showed decellularization of the canine kidney. Our decellularization protocol also preserved the basement membrane of glomerulus, shown by periodic acid methenamine silver, periodic acid-Schiff, fibronectin, and collagen type IV stain. CONCLUSION: Our decellularization protocol could eliminate cellular components and remaining native ECM structures of canine kidney. These results could promote further research into canine kidney regeneration, which may be the first small step to regenerate the canine kidney waiting for renal transplantation.

Characterising keratometry in different dog breeds using an automatic handheld keratometer.

BACKGROUND: Keratometry is clinically important and is routinely performed as part of human ophthalmic examination. In veterinary ophthalmology, little is known about keratometry in dogs, and its practical application has been limited. The present study aimed to describe keratometry in some dog breeds popular in Japan using a handheld keratometer. METHODS: Client-owned dogs of various signalment were enrolled prospectively in the keratometry examination. Interbreed variations in mean corneal curvatures (R1R2avg) and corneal astigmatism (Δ(R1-R2)) were evaluated statistically with respect to their bodyweight based on the data which fulfilled the predetermined inclusion criteria. P<0.05 was considered statistically significant. RESULTS: On examination of 237 dogs from 16 different breeds, R1R2avg (mean±sd) ranged from 7.54±0.30 mm in Pomeranians to 9.28±0.19 mm in golden retrievers. Δ(R1-R2) (mean±sd) ranged from 0.22±0.11 mm in miniature schnauzers to 0.57±0.30 mm in French bulldogs. CONCLUSION: The present study successfully described keratometry in 16 dog breeds. The study revealed considerable interbreed variations in both R1R2avg and Δ(R1-R2), which did not necessarily correlate with bodyweight. These results are useful both clinically in fitting contact lenses in the management of corneal diseases and non-clinically in optometric studies in dogs.

[Hematemesis due to rupture of splenic artery pseudoaneurysm in association with segmental arterial mediolysis:a case report].

A gastric ulcer was detected in a 54-year-old man who underwent upper gastrointestinal endoscopy for hematemesis. An abdominal contrasted computed tomography scan detected a splenic artery aneurysm adjacent to the gastric wall. Endoscopic hemostasis was thought to be risky owing to possible rupture of the aneurysm. Rupture of a splenic artery pseudoaneurysm due to segmental arterial mediolysis (SAM) was diagnosed by abdominal angiography, and subsequently transcatheter arterial embolization was performed. In cases of upper gastrointestinal hemorrhage, hemostasis is often performed during the emergency endoscopic examination. However, in cases of massive gastrointestinal bleeding, the possibility of a splenic artery aneurysm, in association with SAM, should be recognized. The risk of rupturing the aneurysm should be considered in selecting the most suitable treatment.

Transplantation of a decellularized mitral valve complex in pigs.

PURPOSE: Conventional mitral valve replacement is associated with the loss of natural continuity of the mitral valve complex. This study evaluated the morphologic/histological characteristics and function of a decellularized mitral valve used as a transplantable graft. METHODS: Hearts excised from pigs were decellularized by perfusion using detergent. Grafts with the mitral annulus, valve, chordae, and papillary muscle isolated from the decellularized heart were then transplanted into recipient pigs. After transplantation, the function of the graft was analyzed through echocardiography. A histological analysis was performed to evaluate the postoperative features of the decellularized graft. RESULTS: The decellularized graft was successfully transplanted in all cases but one. The remaining grafts maintained their morphology and function. They did not exhibit mitral regurgitation or stenosis. Only one animal survived for 3 weeks, and a histological analysis was able to be performed in this case. The transplanted valve was re-covered with endothelial cells. The microvessels in the papillary muscle were recellularized with vascular endothelial cells, and the papillary muscle was completely attached to the papillary muscle of the recipient. CONCLUSION: The early outcome of decellularized mitral graft transplantation was acceptable. This native organ-derived acellular scaffold is a promising candidate for the replacement of the mitral valve complex.

Decellularized liver scaffolds promote liver regeneration after partial hepatectomy.

The resectable liver volume is strictly limited and this reduces the number of patients who may be treated. Recently, "tissue/organ decellularization", a new approach in bioengineering, has been investigated for its ability to produce a native organ scaffold by removing all the viable cells. Such a scaffold may support the repair of damaged or injured tissue. The purpose of this study was to evaluate the potential contribution of liver scaffolds to hepatic regeneration after hepatectomy. We sutured the partial liver scaffolds onto the surfaces of partially hepatectomized porcine livers and assessed their therapeutic potential by immune histological analysis at various time points. Animals were sacrificed after surgery and the implanted scaffolds were evaluated for the infiltration of various types of cells. Immune histochemical study showed that blood vessel-like structures, covered with CD31 positive endothelial cells and ALB positive cells, were present in all parts of the scaffolds at days 10 and 28. Blood inflow was observed in some of these ductal structures. More interestingly, CK19 and EpCAM positive cells appeared at day 10. These results suggest that the implantation of a decellularized organ scaffold could promote structural reorganization after liver resection.

[Intraductal papillary neoplasm of bile duct developed in a patient with IgG4-related sclerosing cholangitis, autoimmune pancreatitis, and myasthenia gravis].

In the course of treatment for myasthenia gravis, enlargement of a cystic mass in the liver with peripheral bile duct dilation, diffuse pancreatic enlargement, and serum IgG4 level elevation was identified in a 65-year-old man. Following the diagnosis of autoimmune pancreatitis, a left hepatectomy was performed because of suspected malignancy of the cystic lesion. Analysis of the resected specimen revealed the cystic lesion to be a dilated bile duct. Intraductal papillary tumor comprising fibrovascular stalks covered by neoplastic epithelium was identified in the lesion. Infiltration of IgG4-positive plasma cells was discovered around the cystic lesion. Finally, a diagnosis of intraductal papillary neoplasm of bile duct with IgG4-related sclerosing cholangitis was made. Autoimmune diseases, including IgG4-related diseases, require careful observation because of their potential for malignancy.

Efficacy of New Polylactic Acid Nonwoven Fabric as a Hemostatic Agent in a Rat Liver Resection Model.

BACKGROUND: During minimally invasive surgery, efficient and nontoxic hemostats are important for difficult to access bleeding areas. Polylactic acid is an ecofriendly hemostatic agent and we aimed to evaluate the efficacy of a polylactic acid nonwoven fabric (PLAF) developed by Toray Industries, Inc, on liver hemostasis in a preclinical study. MATERIALS AND METHODS: PLAF consists of both 1-µm diameter fibers and 100-µm diameter beaded fibers. Four rats were used, and 2 trough-shaped resections of the liver parenchyma were performed (n = 8 lobes). Immediately after the resection, PLAF (PLAF group: n = 4 lobes) or rayon gauze (Rayon group: n = 4 lobes) were applied on the resected plane and compressed manually. We compared the mean time to hemostasis and blood loss per lobe, as well as histological findings between the groups. RESULTS: The PLAF group had a significantly shorter bleeding time ( P = .006), and showed lower blood loss compared with the Rayon group ( P = .076). Histopathological evaluation showed a large amount of beads on the liver surface in the PLAF group. Aggregated red blood cells evident by electron microscopy and von Willebrand factor immunofluorescence were seen surrounding the beads. The PLAF group showed significantly greater von Willebrand factor expression than the Rayon group ( P = .004). DISCUSSION: This new PLAF showed superior outcomes thanks to its unique characteristic of forming beaded nanofibers, and it has the potential to be an efficient hemostat in minimally invasive surgery in the human body.

Use of ozonated water as a new therapeutic approach to solve current concerns around antitumor treatment.

Tumor hypoxia is a severe problem affecting tumor therapy because it reduces the sensitivity of chemotherapy and radiation therapy. Ozone has been known to improve peripheral blood perfusion and oxygen partial pressure. The effect of ozonated water on tumor hypoxia, alone and in combination with an antitumor drug was studied in the present study. Following intraperitoneal administration of ozonated water to colon-26-bearing mice, the Hoechst 33342-positive area and the intratumoral oxygen partial pressure was significantly increased. The tumor growth rate was more suppressed when ozonated water was combined with cisplatin (CDDP) compared with CDDP treatment alone. The number of Ki-67-positive cells significantly decreased, whereas the number of TUNEL-positive cells significantly increased. The present study showed that ozonated water increased intratumoral blood perfusion and improved tumor hypoxia. In addition, ozonated water increased the therapeutic effect of CDDP. These findings, as well as previous reports, suggest that tumor growth is suppressed after treatment with ozonated water as the amount of CDDP reaching the tumor is increased when the intratumoral blood perfusion is increased due to the ozonated water. Thus, the administration of ozonated water may be a new therapeutic approach to solve current concerns regarding antitumor treatment.