Short-term high-fat and high-carbohydrate diets increase susceptibility to liver injury by inducing hepatic procoagulant and proinflammatory conditions with different balances.

OBJECTIVES: High-fat diet (HFD) and high-carbohydrate diet (HCD) are strongly linked to nonalcoholic fatty liver disease, a hepatic manifestation of metabolic syndrome. The mechanism of pathologic progression from nonalcoholic fatty liver disease to nonalcoholic steatohepatitis, which is a more severe form associated with inflammation and fibrosis, remains poorly understood. Thus, the aim of this study was to investigate and compare the inflammatory and coagulative state of the liver in short-term HFD- or HCD-fed mice with acute liver injury induced by concanavalin A (Con A). METHODS: Histopathologic evaluation, real-time polymerase chain reaction, and immunohistochemical evaluation were performed on the liver of mice fed HFDs and HCDs for 4 d before and after Con A administration. RESULTS: The liver of the HFD-fed mice had larger fibrinogen/fibrin depositions than those fed the HCD. HCD induced the expression of the proinflammatory cytokine tumor necrosis factor-α in the liver. Moreover, the expression of proinflammatory cytokines and chemokines was further enhanced after Con A stimulation in HCD (e.g., interleukin-1α, interleukin-6 at 1 h), with a strong tendency for inflammatory cell infiltration also found (24 h). CONCLUSIONS: Short-term HCD and HFD increased susceptibility to liver injury. HCD tended to induce more intense inflammation, whereas HFD tended to induce more intense hypercoagulation, suggesting that HCD and HFD may have different mechanisms of pathologic progression to nonalcoholic steatohepatitis.

Anorectal Transplantation: The First Long-term Success in a Canine Model.

OBJECTIVE: Anorectal transplantation is a challenging procedure but a promising option for patients with weakened or completely absent anorectal function. SUMMARY BACKGROUND DATA: We constructed a canine model of anorectal transplantation, evaluated the long-term outcomes, and controlled rejection and infection in allotransplantation. METHODS: In the pudendal nerve function study, 6 dogs were randomly divided into 2 groups, transection and anastomosis, and were compared with a control using anorectal manometry, electromyography, and histological examination. In the anorectal transplantation model, 4 dogs were assigned to 4 groups: autotransplant, allotransplant with immunosuppression, allotransplant without immunosuppression, and normal control. Long-term function was evaluated by defecography, videography, and histological examination. RESULTS: In the pudendal nerve function study, anorectal manometry indicated that the anastomosis group recovered partial function 6 months postoperatively. Microscopically, the pudendal nerve and the sphincter muscle regenerated in the anastomosis group. Anorectal transplantation was technically successful with a 3-stage operation: colostomy preparation, anorectal transplantation, and stoma closure. The dog who underwent allotransplantation and immunosuppression had 2 episodes of mild rejection, which were reversed with methylprednisolone and tacrolimus. The dog who underwent allotransplantation without immunosuppression had a severe acute rejection that resulted in graft necrosis. Successful dogs had full defecation control at the end of the study. CONCLUSIONS: We describe the critical role of the pudendal nerve in anorectal function and the first long-term success with anorectal transplantation in a canine model. This report is a proof-of-concept study for anorectal transplantation as a treatment for patients with an ostomy because of anorectal dysfunction.

Cardioprotection via Metabolism for Rat Heart Preservation Using the High-Pressure Gaseous Mixture of Carbon Monoxide and Oxygen.

The high-pressure gas (HPG) method with carbon monoxide (CO) and oxygen (O2) mixture maintains the preserved rat heart function. The metabolites of rat hearts preserved using the HPG method (HPG group) and cold storage (CS) method (CS group) by immersion in a stock solution for 24 h were assessed to confirm CO and O2 effects. Lactic acid was significantly lower and citric acid was significantly higher in the HPG group than in the CS group. Moreover, adenosine triphosphate (ATP) levels as well as some pentose phosphate pathway (PPP) metabolites and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were significantly higher in the HPG group than in the CS group. Additionally, reduced glutathione (GSH), which protects cells from oxidative stress, was also significantly higher in the HPG group than in the CS group. These results indicated that each gas, CO and O2, induced the shift from anaerobic to aerobic metabolism, maintaining the energy of ischemic preserved organs, shifting the glucose utilization from glycolysis toward PPP, and reducing oxidative stress. Both CO and O2 in the HPG method have important effects on the ATP supply and decrease oxidative stress for preventing ischemic injury. The HPG method may be useful for clinical application.

Developing spray-freeze-dried particles containing a hyaluronic acid-coated liposome-protamine-DNA complex for pulmonary inhalation.

The liposome-protamine-DNA complex (LPD) is an effective cationic carrier of various nucleic acid constructs such as plasmid DNA and small interfering RNA (siRNA). Hyaluronic acid coated on LPD (LPDH) reduces cytotoxicity and maintains the silencing effect of LPD-encapsulated siRNA. Herein, we aim to develop LPD- or LPDH-containing spray-freeze-dried particles (SFDPs) for therapeutic delivery of siRNA to the lungs. LPD- or LPDH-containing SFDPs (LPD- or LPDH-SFDPs) were synthesized and their structure and function as gene carriers were evaluated using physical and biological methods. The particle size of LPDH, but not of LPD, was constant after re-dispersal from the SFDPs and the amount of siRNA encapsulated in LPDH was larger than that in LPD after re-dispersal from the SFDPs. The in vitro pulmonary inhalation properties of LPDH-SFDPs and LPD-SFDPs were almost the same. The cytotoxicity of LPDH-SFDPs in human umbilical vein endothelial cells (HUVEC) was greatly decreased compared with that of LPD-SFDPs. In addition, Bcl-2 siRNA in LPDH-SFDPs had a significant gene silencing effect in human lung cancer cells (A549), whereas Bcl-2 siRNA in LPD-SFDPs had little effect. These results indicate that compared with LPD, LPDH is more useful for developing SFDPs for siRNA pulmonary inhalation.

The effectiveness of traditional Japanese medicine Goshajinkigan in irradiation-induced aspermatogenesis in mice.

BACKGROUND: Infertility and gonadal dysfunction are well known side-effects by cancer treatment in males. In particularly, chemotherapy and radiotherapy induced testicular damage, resulting in prolonged azoospermia. However, information regarding therapeutics to treat spermatogenesis disturbance after cancer treatment is scarce. Recently, we demonstrated that Goshajinkigan, a traditional Japanese medicine, can completely rescue severe busulfan-induced aspermatogenesis in mice. In this study, we aimed to detect the effects of Goshajinkigan on aspermatogenesis after irradiation. METHODS: This is animal research about the effects of traditional Japanese medicine on infertility after cancer treatment. C57BL/6 J male mice received total body irradiation (TBI: a single dose of 6Gy) at 4 weeks of age and after 60 days were reared a Goshajinkigan (TJ107)-containing or TJ107-free control diet from day 60 to day 120. Then, two untreated females were mated with a single male from each experimental group. On day 60, 120 and 150, respectively, the sets of testes and epididymis of the mice in each group after deep anesthetization were removed for histological and cytological examinations. RESULTS: Histological and histopathological data showed that 6Gy TBI treatment decreased the fertility rate (4/10) in the control diet group; in contrast, in the TJ107-diet group, the fertility rate was 10/10 (p < 0.05 vs. 6Gy group). Supplementation with TJ107 was found to rescue the disrupted inter-Sertoli tight junctions via the normalization of claudin11, occludin, and ZO-1 expression and reduce serum anti-germ cell autoantibodies. CONCLUSIONS: These findings show the therapeutic effect on TBI-induced aspermatogenesis and the recovering disrupted gonadal functions by supplementation with TJ107.

Spraying urea solution reduces formaldehyde levels during gross anatomy courses.

Formaldehyde (FA) is frequently used to embalm human cadavers that are employed to teach gross anatomy to medical and dental students. However, exposure to FA is harmful to both students and educators. The aim of this study was to reduce the FA levels in the anatomy dissection hall by spraying an FA scavenger solution. We measured the changes in FA levels after administering FA scavenger solutions to liquid, wet paper towels, organs, and cadavers containing FA. Among L-cysteine, N-ethyl urea, and urea, the latter was found to have the strongest scavenging power towards the FA in the liquid. The molar concentration of urea that most efficiently reduced the levels of volatilized FA from the wet paper towels was the same as that of the FA. After spraying the urea solution, the volatilized FA levels immediately decreased, reaching their minimum at 60 min, and remained low even after 240 min. Spraying the urea solution onto the organs reduced the levels of FA volatilized from the surfaces of organs but not those from the insides of the organs. In the dissection hall used for the gross anatomy course at Tokyo Medical University, the FA levels were significantly decreased after spraying the urea solution onto the cadavers. Moreover, dissection could be performed without the cadavers putrefying during the 4-month course. These results indicate that various institutes could use urea solution spray to effectively reduce the FA levels in the dissection hall and thus ensure the safety of students and educators.

Pathological effect of arterial ischaemia and venous congestion on rat testes.

Many studies on various organs have concluded that venous congestion (VC) causes severe organ dysfunction with elevation of oxidative stress relative to that of arterial ischaemia (AI). However, a comparison of the pathological effects of AI and VC on the testes has not been conducted. In this study, models of AI and VC and their reperfusion in rat testes, respectively, were developed and analysed. Testicular arteries or veins were interrupted for 6 h, re-perfused and kept for 4 weeks; the effects on the testes were then evaluated. Severe spermatogenic disturbances were observed at 4 weeks after reperfusion in AI but not in VC. At 6 h after blood flow interruption, oxidative stress was significantly increased and germ cells were severely damaged in AI compared with those in VC. RT-PCR analyses revealed that haem oxygenase-1, which exhibits anti-oxidative effects, and vascular endothelial growth factor-A, which exhibits vasculogenic effects, were significantly increased in VC but not in AI. Surprisingly, the results of our experiment in rat testes differed from those of experiments in previous studies performed in other organs. Oxidative stress in testes was more easily elevated by AI than it was by VC, explainable by the different experimental conditions.

High-pressure carbon monoxide preserves rat kidney grafts from apoptosis and inflammation.

Renal ischemia-reperfusion (I/R) injury is unavoidable in kidney transplantation (KTx) and frequently influences both short- and long-term allograft survival. Carbon monoxide (CO) has attracted attention as a medical gas with anti-inflammatory and anti-apoptotic effects. We investigated a new strategy for organ preservation using ex vivo application of high-pressure CO in an experimental rat KTx model. We preserved kidney grafts using a high-pressure chamber filled with mixed gases composed of CO and O2. We found that cold I/R injury resulted in progressive deterioration of renal graft function in University of Wisconsin solution, whereas CO significantly improved renal function. We confirmed that CO decreased oxidative stress and mRNA expression of proinflammatory cytokines and inhibited tubular apoptosis in the early phases. Western blot analysis demonstrated that CO increased phosphatidylinositol-3 kinase and phosphorylation of Akt and p38 mitogen-activated protein kinase. Furthermore, CO significantly alleviated tubular injury scores and suppressed the development of interstitial fibrosis at 100 days after KTx. Thus, high-pressure mixed CO and O2 gases successfully preserved rat kidney grafts for 24 h by protecting tubular epithelial cells from apoptosis and inhibiting inflammation.

Specific autoantigens identified by sera obtained from mice that are immunized with testicular germ cells alone.

There are various autoimmunogenic antigens (AIs) in testicular germ cells (TGCs) recognized as foreign by the body's immune system. However, there is little information of TGC-specific AIs being available. The aim of this study is to identify TGC-specific AIs. We have previously established that immunization using viable syngeneic TGC can also induce murine experimental autoimmune orchitis (EAO) without using any adjuvant. This study is to identify TGC-specific AIs by TGC liquid chromatography-tandem mass spectrometry analysis, followed by two-dimensional gel electrophoresis that reacted with serum IgG from EAO mice. In this study, we identified 11 TGC-specific AIs that reacted with serum from EAO mice. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs were significantly higher in only mature testis compared to other organs. Moreover, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies.

History and future of human cadaver preservation for surgical training: from formalin to saturated salt solution method.

Traditionally, surgical training meant on-the-job training with live patients in an operating room. However, due to advancing surgical techniques, such as minimally invasive surgery, and increasing safety demands during procedures, human cadavers have been used for surgical training. When considering the use of human cadavers for surgical training, one of the most important factors is their preservation. In this review, we summarize four preservation methods: fresh-frozen cadaver, formalin, Thiel's, and saturated salt solution methods. Fresh-frozen cadaver is currently the model that is closest to reality, but it also presents myriad problems, including the requirement of freezers for storage, limited work time because of rapid putrefaction, and risk of infection. Formalin is still used ubiquitously due to its low cost and wide availability, but it is not ideal because formaldehyde has an adverse health effect and formalin-embalmed cadavers do not exhibit many of the qualities of living organs. Thiel's method results in soft and flexible cadavers with almost natural colors, and Thiel-embalmed cadavers have been appraised widely in various medical disciplines. However, Thiel's method is relatively expensive and technically complicated. In addition, Thiel-embalmed cadavers have a limited dissection time. The saturated salt solution method is simple, carries a low risk of infection, and is relatively low cost. Although more research is needed, this method seems to be sufficiently useful for surgical training and has noteworthy features that expand the capability of clinical training. The saturated salt solution method will contribute to a wider use of cadavers for surgical training.

Busulfan pretreatment for transplantation of rat spermatogonia differentially affects immune and reproductive systems in male recipient mice.

Testicular cell transplantation has generally been performed by using immune-deficient recipient mice to investigate the biology of spermatogonial stem cells (SSCs), the production of transgenic animals, and restoration of fertility. Recently, we demonstrated that rat spermatogenesis can occur in the seminiferous tubules of immune-competent recipient mice via pretreatment with busulfan (Myleran, 1, 4-butanediol methanesulfonate, 40 mg/kg) after transplantation of rat SSCs. However, considering the immunosuppressive effect of busulfan, there is a possibility that busulfan itself causes immune suppression in immune-competent recipient mice. The aim of this study was to determine the effects of busulfan on the immune system and spermatogenesis in immune-competent recipient mice. The results showed that at 60 days after busulfan treatment, just the same time as the transplantation, the recovery could be seen in the immune system including cell counts and functions of T and B lymphocytes in the spleen, but the spermatogenesis was more compromised. This study demonstrated that after busulfan pretreatment the immune system in immune-competent recipient mice had recovered by the time that rat spermatogenesis could occur in the murine testis. It became clear that xenogenic spermatogenesis can be tolerated in seminiferous tubules in the testes of immune-competent mice.

Saturated salt solution method: a useful cadaver embalming for surgical skills training.

This article evaluates the suitability of cadavers embalmed by the saturated salt solution (SSS) method for surgical skills training (SST). SST courses using cadavers have been performed to advance a surgeon's techniques without any risk to patients. One important factor for improving SST is the suitability of specimens, which depends on the embalming method. In addition, the infectious risk and cost involved in using cadavers are problems that need to be solved. Six cadavers were embalmed by 3 methods: formalin solution, Thiel solution (TS), and SSS methods. Bacterial and fungal culture tests and measurement of ranges of motion were conducted for each cadaver. Fourteen surgeons evaluated the 3 embalming methods and 9 SST instructors (7 trauma surgeons and 2 orthopedists) operated the cadavers by 21 procedures. In addition, ultrasonography, central venous catheterization, and incision with cauterization followed by autosuture stapling were performed in some cadavers. The SSS method had a sufficient antibiotic effect and produced cadavers with flexible joints and a high tissue quality suitable for SST. The surgeons evaluated the cadavers embalmed by the SSS method to be highly equal to those embalmed by the TS method. Ultrasound images were clear in the cadavers embalmed by both the methods. Central venous catheterization could be performed in a cadaver embalmed by the SSS method and then be affirmed by x-ray. Lungs and intestines could be incised with cauterization and autosuture stapling in the cadavers embalmed by TS and SSS methods. Cadavers embalmed by the SSS method are sufficiently useful for SST. This method is simple, carries a low infectious risk, and is relatively of low cost, enabling a wider use of cadavers for SST.

Contribution of IL-12/IL-35 common subunit p35 to maintaining the testicular immune privilege.

The testis is an organ with immune privilege. The comprehensive blood-testis barrier formed by Sertoli cells protects autoimmunogenic spermatozoa and spermatids from attack by the body's immune system. The interleukin (IL)-6/IL-12 family cytokines IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Epstein-Barr virus-induced gene 3 [EBI3]), and IL-35 (p35/EBI3) play critical roles in the regulation of various immune responses, but their roles in testicular immune privilege are not well understood. In the present study, we investigated whether these cytokines are expressed in the testes and whether they function in the testicular immune privilege by using mice deficient in their subunits. Expression of EBI3 was markedly increased at both mRNA and protein levels in the testes of 10- or 12-week-old wild-type mice as compared with levels in 2-week-old mice, whereas the mRNA expression of p40 was markedly decreased and that of p35 was conserved between these two groups. Lack of EBI3, p35, and IL-12 receptor ß2 caused enhanced infiltration of lymphocytes into the testicular interstitium, with increased interferon-γ expression in the testes and autoantibody production against mainly acrosomal regions of spermatids. Spermatogenic disturbance was more frequently observed in the seminiferous tubules, especially when surrounded by infiltrating lymphocytes, of these deficient mice than in those of wild-type mice. In particular, p35-deficient mice showed the most severe spermatogenic disturbance. Immunohistochemical analyses revealed that endothelial cells and peritubular cells in the interstitium were highly positive for p35 at both ages, and CD163+ resident macrophages positive for p35 and EBI3, possibly producing IL-35, were also detected in the interstitium of 12-week-old mice but not those of 2-week-old mice. These results suggest that p35 helps in maintaining the testicular immune privilege, in part in an IL-35-dependent manner.

Neonatal estrogen treatment with ß-estradiol 17-cypionate induces in post-pubertal mice inflammation in the ductuli efferentes, epididymis, and vas deferens, but not in the testis, provoking obstructive azoospermia.

Neonatal estrogen treatment (NET) induces morphological changes in male reproductive organs. NET with ß-estradiol 17-cypionate is reported to induce inflammation with stromal-epithelial abnormalities in the prostate and seminal vesicles in post-pubertal mice. The aim of this study was to investigate the histopathology of the testis, ductuli efferentes, epididymis, and vas deferens in mice after NET with ß-estradiol 17-cypionate. No morphological changes in these organs were observed until 4 weeks after NET. However, some inflammatory cells were found in epididymis and vas deferens 6 weeks after NET. Eight weeks after NET, inflammatory cells spread to the ductuli efferentes and inflammation was severe from 6 to 12 weeks after NET. Inflammatory cells were never seen in the whole testis, but cystic dilatation of the rete testes with spermatogenic disturbance was found around the mediastinum testis. Many inflammatory cells emigrated into the lumen of the epididymis, resulting in complete absence of spermatozoa in the vas deferens. Most of the inflammatory cells penetrating into the epithelial layers of epididymal ducts were neutrophils. These results indicate that in post-pubertal mice, NET with ß-estradiol 17-cypionate induces inflammation in the ductuli efferentes, epididymis, and vas deferens, but not in the testis, provoking obstructive azoospermia.

The effects of adjuvants on autoimmune responses against testicular antigens in mice.

Experimental autoimmune orchitis (EAO) is a model of immunologic male infertility and pathologically characterized by lymphocytic inflammation, which causes breakdown of the testicular immune privilege with spermatogenic disturbance. Generally, murine EAO is induced by immunization with testicular homogenate (TH) from the testes of donor mice + complete Freund's adjuvant (CFA) + Bordetella pertussigens (BP), and it has been considered that treatment with these two adjuvants is required to enhance the immune response against testicular antigens. However, there remains a possibility that CFA and BP may affect autoimmune responses against the testicular antigens without TH. In the present study, we examined this possibility using real-time RT-PCR, Western blotting and immunohistochemical staining. The results demonstrated that immunization with TH in combination with CFA and BP evoked more severe EAO than that with only TH. Real-time RT-PCR analyses revealed that Fas mRNA expression in TH+CFA+BP-induced EAO was significantly higher than that in TH-induced EAO. Interestingly, IL-6 mRNA expression dramatically increased in TH+CFA+BP-induced EAO; however, no apparent change in IL-6 mRNA expression occurred in TH-induced EAO. It was also noted that treatment with CFA and BP alone augmented autoimmune reactions against some testicular autoantigens. These results indicates that these adjuvants are helpful in evoking severe EAO, and treatment with the adjuvants alone can evoke autoimmune reactions against some testicular autoantigens despite the use of no TH.

Hydrogen-rich University of Wisconsin solution attenuates renal cold ischemia-reperfusion injury.

BACKGROUND: Renal ischemia-reperfusion (I/R) injury is unavoidable in kidney transplantation and frequently influences both short- and long-term allograft survival rates. One of the major events in I/R injury is the generation of cytotoxic oxygen radicals. Recently, hydrogen gas has been reported to display antioxidant properties and protective effects against organ dysfunction induced by various I/R injuries. We investigated whether hydrogen-rich University of Wisconsin (HRUW) solution attenuates renal cold I/R injury. METHODS: We prepared HRUW solution by a novel method involving immersion of centrifuge tubes containing UW solution into hydrogen-saturated water. Hydrogen readily permeates through the centrifuge tubes, and thus, the hydrogen concentration of the UW solution gradually increases in a time-dependent manner. Syngeneic rat kidney transplantation was performed, and the animals were divided into three groups: recipients with nonpreserved grafts (control group), recipients with grafts preserved in UW solution for 24 to 48 hr (UW group), and recipients with grafts preserved in HRUW solution for 24 to 48 hr (HRUW group). RESULTS: In the early phases, HRUW solution decreased oxidative stress, tubular apoptosis, and interstitial macrophage infiltration in the kidney grafts. Consequently, HRUW solution improved renal function and prolonged recipient survival rate compared with simple cold storage using UW solution. Histopathologically, HRUW treatment alleviated tubular injury and suppressed development of interstitial fibrosis. CONCLUSIONS: HRUW solution improved graft function and prolonged graft survival compared with simple cold storage using UW solution by protecting tubular epithelial cells from inflammation and apoptosis. Our new method of organ preservation is a groundbreaking, safe, and simple strategy that may be applied in the clinical setting.

Seventy-Two-Hour Preservation, Resuscitation, and Transplantation of an Isolated Rat Heart with High Partial Pressure Carbon Monoxide Gas (PCO = 400 hPa) and High Partial Pressure Carbon Dioxide (PCO2 = 100 hPa).

The cardiac cavity of an isolated rat heart was filled with a Krebs-Henseleit (KH) solution, and the heart was hung in a high-pressure chamber. After the high-pressure chamber had been filled with a mixed gas (PCO = 400 hPa, PCO2 = 100 hPa, PO2 = 900 hPa, PHe = 5600 hPa) and preserved for 72 h, we performed a cervical ectopic heart transplantation on a recipient rat and resuscitated the preserved heart. This is the first incidence in the world of a mammalian organ having been successfully preserved and resuscitated after 72 h via a desiccation method.

Seventy-two-hour preservation, resuscitation, and transplantation of an isolated rat heart with high partial pressure carbon monoxide gas (PCO = 400 hPa) and high partial pressure carbon dioxide (PCO(2) = 100 hPa).

The cardiac cavity of an isolated rat heart was filled with a Krebs-Henseleit (KH) solution, and the heart was hung in a high-pressure chamber. After the high-pressure chamber had been filled with a mixed gas (PCO = 400 hPa, PCO(2) = 100 hPa, PO(2) = 900 hPa, PHe = 5600 hPa) and preserved for 72 h, we performed a cervical ectopic heart transplantation on a recipient rat and resuscitated the preserved heart. This is the first incidence in the world of a mammalian organ having been successfully preserved and resuscitated after 72 h via a desiccation method.