Involvement of Degenerating 21.5 kDa Isoform of Myelin Basic Protein in the Pathogenesis of the Relapse in Murine Relapsing-Remitting Experimental Autoimmune Encephalomyelitis and MS Autopsied Brain.

Multiple sclerosis (MS) is the chronic inflammatory demyelinating disease of the CNS. Relapsing-remitting MS (RRMS) is the most common type of MS. However, the mechanisms of relapse and remission in MS have not been fully understood. While SJL mice immunized with proteolipid protein (PLP) develop relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE), we have recently observed that some of these mice were resistant to the active induction of relapsing EAE after initial clinical and histological symptoms of EAE with a severity similar to the relapsing EAE mice. To clarify the mechanism of relapsing, we examined myelin morphology during PLP139-151-induced RR-EAE in the SJL mice. While RR-EAE mice showed an increased EAE severity (relapse) with CNS inflammation, demyelination with abnormal myelin morphology in the spinal cord, the resistant mice exhibited a milder EAE phenotype with diminished relapse. Compared with the RR-EAE mice, the resistant mice showed less CNS inflammation, demyelination, and abnormalities of the myelin structure. In addition, scanning electron microscopic (SEM) analysis with the osmium-maceration method displayed ultrastructural abnormalities of the myelin structure in the white matter of the RR-EAE spinal cord, but not in that of the resistant mice. While the intensity of myelin staining was reduced in the relapsing EAE spinal cord, immunohistochemistry and immunoblot analysis revealed that the 21.5 kDa isoform of degenerating myelin basic protein (MBP) was specifically induced in the relapsing EAE spinal cord. Taken together, the neuroinflammation-induced degenerating 21 kDa isoform of MBP sheds light on the development of abnormal myelin on the relapse of MS pathogenesis.

Association Between Workplace Social Support and Use of Health-Promoting Wearable Devices: A Prospective Cohort Study of Japanese Employees.

We examined the relationship between workplace environmental factors, including support from supervisors and colleagues, and the continued use of a wearable device meant to promote occupational health. One hundred employees at a Japanese manufacturing company participated in a 3-month study, and information related to their physical health status was recorded by a wearable device. We analyzed the results using the χ2 test and logistic regression analysis. We found that men aged 40-49 years and employees reporting low support from supervisors and colleagues were significantly more likely to be continuing device users. Participants with low workplace support had adjusted odds ratios approximately two to three times higher than those with high levels of support, which was significant. Employees with low workplace support were able to communicate at work, access appropriate support, and enthusiastically participate in occupational health promotion with little psychological difficulty in using the device. Occupational health promotion using wearable devices can complement traditional face-to-face occupational health promotion.

Ultrastructural Features of the Aortic Wall in a Patient with Kommerell Diverticulum.

We report on the ultrastructural features of the aortic wall in a patient with Kommerell diverticulum. A 70-year-old woman with a right aortic arch, aberrant left subclavian artery, and Kommerell diverticulum underwent a successful total arch replacement plus the frozen elephant trunk procedure with anatomical left subclavian artery reconstruction. Small pieces of the ascending aorta, distal arch, right common carotid artery, and left subclavian artery were investigated ultrastructurally. In the ascending aortic wall, multiple cystic cavities were observed in the subintimal region of the media by scanning electron microscopy. Changes in organelles, including mild dilation of rough-surfaced endoplasmic reticulum and mitochondrial swelling and degrading, were also observed in all specimens by transmission electron microscopy. These ultrastructural features may indicate the fragility or stress of the aortic wall and are useful when considering the early surgical intervention of a patient with Kommerell diverticulum.

Kallikrein 6 secreted by oligodendrocytes regulates the progression of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), and experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of the disease. Here, we examined the pathophysiological role of Kallikrein 6 (Klk6), a serine protease produced by oligodendrocytes (OLs), in EAE using Klk6 knockout (Klk6-/-) mice. Compared with Klk6+/+ (wild-type) mice, Klk6-/- mice showed milder EAE symptoms, including delayed onset and milder paralysis. Loss of Klk6 suppressed matrix metalloprotease-9 expression and diminished the infiltration of peripheral inflammatory cells into the CNS by decreasing blood-brain barrier (BBB) permeability and reducing expression levels of inflammatory cytokines, chemokines and their receptors. Scanning electron microscopic analysis revealed demyelination characterized by myelin detachment from the axons in the early phase of EAE progression (days 3-7) in Klk6+/+ mice but not in Klk6-/- mice. Interestingly, anti-MOG (myelin oligodendrocyte glycoprotein) autoantibody was also detected in the cerebrospinal fluid (CSF) and spinal cord on day 3 after MOG immunization. Furthermore, treatment of primary cultured OLs with anti-MOG autoantibody induced oligodendroglial morphological changes and increases in myelin basic protein and Klk6 expression. We also developed a novel enzyme-linked immunoabsorbent assay method for detecting activated KLK6 in human CSF. In human autopsy brain samples, expression of active KLK6 was detected in OLs using an antibody that specifically recognizes the protein's activated form. Taken together, our findings demonstrate that Klk6 secreted by OLs plays a critical role in the pathogenesis of EAE/MS and that it might serve as a potential therapeutic target for MS.

Multiple sorting systems for secretory granules ensure the regulated secretion of peptide hormones.

Prior to secretion, regulated peptide hormones are selectively sorted to secretory granules (SGs) at the trans-Golgi network (TGN) in endocrine cells. Secretogranin III (SgIII) appears to facilitate SG sorting process by tethering of protein aggregates containing chromogranin A (CgA) and peptide hormones to the cholesterol-rich SG membrane (SGM). Here, we evaluated the role of SgIII in SG sorting in AtT-20 cells transfected with small interfering RNA targeting SgIII. In the SgIII-knockdown cells, the intracellular retention of CgA was greatly impaired, and only a trace amount of CgA was localized within the vacuoles formed in the TGN, confirming the significance of SgIII in both the tethering of CgA-containing aggregates and the establishment of the proper SG morphology. Although the intracellular retention of proopiomelanocortin (POMC) was considerably impaired in SgIII-knockdown cells, residual adrenocorticotropic hormone (ACTH)/POMC was still localized to some few remaining SGs together with another granin protein, secretogranin II (SgII), and was secreted in a regulated manner. Biochemical analyses indicated that SgII bound directly to the SGM in a cholesterol-dependent manner and was able to retain the aggregated form of POMC, revealing a latent redundancy in the SG sorting and retention mechanisms, that ensures the regulated secretion of bioactive peptides.

Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction.

Disruption of epithelial barrier function was identified as one of the pathologic mechanisms in inflammatory bowel diseases (IBD). Epithelial barrier consists of various intercellular junctions, in which the tight junction (TJ) is an important component. However, the regulatory mechanism of tight junction is still not clear. Here we examined the role of focal adhesion kinase (FAK) in the epithelial barrier function on Caco-2 monolayers using a specific FAK inhibitor, PF-573, 228 (PF-228). We found that the decrease of transepithelial resistance and the increase of paracellular permeability were accompanied with the inhibition of autophosphorylation of FAK by PF-228 treatment. In addition, PF-228 inhibited the FAK phosphorylation at Y576/577 on activation loop by Src, suggesting Src-dependent regulation of FAK in Caco-2 monolayers. In an ethanol-induced barrier injury model, PF-228 treatment also inhibited the recovery of transepithelial resistance as well as these phosphorylations of FAK. In a sucrose gradient ultracentrifugation, FAK co-localized with claudin-1, an element of the TJ complex, and they co-migrate after ethanol-induced barrier injury. Immunofluorescence imaging analysis revealed that PF-228 inhibited the FAK redistribution to the cell border and reassembly of TJ proteins in the recovery after ethanol-induced barrier injury. Finally, knockdown of FAK by siRNA resulted in the decrease of transepithelial resistance. These findings reveal that activation of FAK is necessary for maintaining and repairing epithelial barrier in Caco-2 cell monolayer via regulating TJ redistribution.

Nerve growth factor stimulates regeneration of perivascular nerve, and induces the maturation of microvessels around the injured artery.

An immature vasa vasorum in the adventitia of arteries has been implicated in induction of the formation of unstable atherosclerotic plaques. Normalization/maturation of the vasa vasorum may be an attractive therapeutic approach for arteriosclerotic diseases. Nerve growth factor (NGF) is a pleotropic molecule with angiogenic activity in addition to neural growth effects. However, whether NGF affects the formation of microvessels in addition to innervation during pathological angiogenesis is unclear. In the present study, we show a new role for NGF in neovessels around injured arterial walls using a novel in vivo angiogenesis assay. The vasa vasorum around arterial walls was induced to grow using wire-mediated mouse femoral arterial injury. When collagen-coated tube (CCT) was placed beside the injured artery for 7-14 days, microvessels grew two-dimensionally in a thin layer on the CCT (CCT-membrane) in accordance with the development of the vasa vasorum. The perivascular nerve was found at not only arterioles but also capillaries in the CCT-membrane. Biodegradable hydrogels containing VEGF and NGF were applied around the injured artery/CCT. VEGF significantly increased the total length and instability of microvessels within the CCT-membrane. In contrast, NGF induced regeneration of the peripheral nerve around the microvessels and induced the maturation and stabilization of microvessels. In an ex vivo nerve-free angiogenesis assay, although NGF potentially stimulated vascular sprouting from aorta tissues, no effects of NGF on vascular maturation were observed. These data demonstrated that NGF had potent angiogenic effects on the microvessels around the injured artery, and especially induced the maturation/stabilization of microvessels in accordance with the regeneration of perivascular nerves.

Important Constituents of Heavy Water-containing Solution for Cold Storage and Subsequent Reperfusion on an Isolated Perfused Rat Liver.

The University of Wisconsin (UW) solution is the most effective preservation solution currently used; however, to safely use expanded-criteria donor grafts, a new cold storage solution that alleviates graft injury more effectively is required. We prepared a heavy water (D2O)-containing buffer, Dsol, and observed strong protective effects during extended cold storage of rat hearts and livers. In the current study, we modified Dsol (mDsol) and tested its efficacy. The aim of the present study was to determine whether mDsol could protect the rat liver more effectively than the UW solution and to clarify the roles of D2O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D2O or DFX (mDsol-D2O[-], mDsol-DFX[-]), and subsequently reperfused on an isolated perfused rat liver for 90 minutes at 37°C. In the UW group, the liver was dehydrated during cold storage and rapidly expanded during reperfusion. Accordingly, the cumulative weight change was the highest in the UW group, together with augmented portal veinous resistance and ALT leakage and decreased oxygen consumption rate and bile production. These changes were significantly suppressed in the mDsol-treated group. In the mDsol-D2O(-) and mDsol-DFX(-) groups offered partial protection. In conclusion, mDsol appeared to be superior to the UW solution for simple cold storage of the rat liver, presumably due to improved microcirculation in the early phase of reperfusion. Both heavy water and deferoxamine are essential for alleviating seamless organ swelling that occurs during cold storage and subsequent reperfusion.

Machine perfusion preservation with hemoglobin based oxygen vesicles alleviate ultrastructural damages in porcine liver donated after cardiac death.

Midthermic machine perfusion (MMP) of post-circulatory arrest donor liver grafts has the advantage of preserving the functional ultrastructure of hepatocytes in donor grafts. It was reported that oxygenation during MMP reduces portal venous resistance and increases bile production. The MMP with hemoglobin-based oxygen vesicles (HbV) keeps the lower aspartate aminotransferase level (an indicator of liver injury) and maintains the functional ultrastructure of mitochondria in the hepatocytes. To evaluated differences of ultrastructural damages in donor livers between the MMP with and without HbV, porcine liver grafts after 60 min of warm ischemia were perfused at 22°C for 4 h with or without HbV, and a part of liver grafts were analyzed by transmission electron microscopy (TEM) and osmium-maceration scanning electron microscopy (OM-SEM). The remaining grafts were perfused with autologous blood at 38°C for 2 h in an isolated liver reperfusion model (IRM) that mimics the inside of the body after transplantation, and then analyzed by TEM and OM-SEM. Hepatocytes after MMP had small round mitochondria with rod-shaped cristae and reticulovesicular rough endoplasmic reticulum (rER) in both HbV(+) and HbV(-) livers. After IRM of HbV(+) livers, the well-developed lamellar rER was often found in hepatocytes. Liver sinusoidal endothelial cells (LSECs) after MMP contained some large vacuolar structures containing amorphous garbage in the cytoplasm, and their size along with appearance frequency were smaller and lower, respectively, in HbV(+) livers than HbV(-). Oxygenation during the MMP by using HbV suppressed the ultrastructural damages in donor livers, in particular for the LSECs. RESEARCH HIGHLIGHTS: Liver sinusoidal endothelial cells after midthermic machine perfusion had large vacuolar organelles with amorphous garbage. Oxygenation during the perfusion made them less and smaller, ultrastructurally supporting its utility.

Exploration of Optimal pH in Hypothermic Machine Perfusion for Rat Liver Grafts Retrieved after Circulatory Death.

Ex vivo hypothermic machine perfusion (HMP) is a strategy for controlling ischemia-reperfusion injury in donation after circulatory death (DCD) liver transplantation. The pH of blood increases with a decrease in temperature and water dissociation, leading to a decrease in [H+]. This study aimed to verify the optimal pH of HMP for DCD livers. Rat livers were retrieved 30 min post-cardiac arrest and subjected to 3-h cold storage (CS) in UW solution (CS group) or HMP with UW-gluconate solution (machine perfusion [MP] group) of pH 7.4 (original), 7.6, 7.8, and 8.0 (MP-pH 7.6, 7.8, 8.0 groups, respectively) at 7-10 °C. The livers were subjected to normothermic perfusion to simulate reperfusion after HMP. All HMP groups showed greater graft protection compared to the CS group due to the lower levels of liver enzymes in the former. The MP-pH 7.8 group showed significant protection, evidenced by bile production, diminished tissue injury, and reduced flavin mononucleotide leakage, and further analysis by scanning electron microscopy revealed a well-preserved structure of the mitochondrial cristae. Therefore, the optimum pH of 7.8 enhanced the protective effect of HMP by preserving the structure and function of the mitochondria, leading to reduced reperfusion injury in the DCD liver.

Combination of Cold Storage in a Heavy Water-Containing Solution and Post-Reperfusion Hydrogen Gas Treatment Reduces Ischemia-Reperfusion Injury in Rat Livers.

We previously reported the efficacy of cold storage (CS) using a heavy water-containing solution (Dsol) and post-reperfusion hydrogen gas treatment separately. This study aimed to clarify the combined effects of these treatments. Rat livers were subjected to 48-hour CS and a subsequent 90-minute reperfusion in an isolated perfused rat liver system. The experimental groups were the immediately reperfused control group (CT), the CS with University of Wisconsin solution (UW) group, the CS with Dsol group, the CS with UW and post-reperfusion H2 treatment group (UW-H2), and the CS with Dsol and post-reperfusion H2 group (Dsol-H2). We first compared the Dsol-H2, UW, and CT groups to evaluate this alternative method to conventional CS. The protective potential of the Dsol-H2 group was superior to that of the UW group, as evidenced by lower portal venous resistance and lactate dehydrogenase leakage, a higher oxygen consumption rate, and increased bile production. Multiple comparison tests among the UW, Dsol, UW-H2, and Dsol-H2 groups revealed that both treatments, during CS and after reperfusion, conferred a similar extent of protection and showed additive effects in combination therapy. Furthermore, the variance in all treatment groups appeared smaller than that in the no-treatment or no-stress groups, with excellent reproducibility. In conclusion, combination therapy with Dsol during CS and hydrogen gas after reperfusion additively protects against graft injury.

Combined Use of Subnormothermic Extracorporeal Support and Hypothermic Oxygenated Machine Perfusion for Liver Graft After Cardiac Death in Pigs.

BACKGROUND: The use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. This study aims to determine the benefits of extracorporeal membrane oxygenation (ECMO) and hypothermic oxygenated machine perfusion (HOPE) in a large animal model of DCD liver. METHODS: After cardiac arrest, the abdominal aorta and the inferior vena cava were cannulated and connected to an ECMO circuit. Porcine livers were perfused in situ with ECMO at 22°C for 60 minutes after 45 minutes of cardiac death. Then, the livers were perfused for 4 hours by cold storage (CS) or HOPE. In group 1, non-in situ ECMO and grafts were preserved by HOPE. In group 2, in situ ECMO and grafts were preserved by HOPE. In group 3, in situ ECMO and grafts were preserved by CS. After preservation, all grafts were evaluated using an isolated reperfusion model (IRM) with autologous blood for 2 hours. RESULTS: During HOPE, aspartate aminotransferase (AST) levels and hepatic arterial pressure in group 2 tended to be lower than in group 1. Hematoxylin-eosin staining findings after HOPE showed more massive sinusoidal congestion and hepatocyte cytoplasmic vacuolization in group 1 than in group 2. The AST and LDH levels in group 2 at the start-up of IRM tended to be lower than in group 1. CONCLUSIONS: The combined use of in situ subnormothermic ECMO and HOPE is essential for the functional recovery of DCD liver grafts.

Beneficial Effects of Combined Use of Extracorporeal Membrane Oxygenation and Hypothermic Machine Perfusion in Porcine Donors after Cardiac Death for Liver Transplantation.

Grafts from donors after cardiac death (DCD) have greatly contributed to expanding the donor organ pool. This study aimed to determine the benefits of subnormothermic extracorporeal membrane oxygenation (ECMO) and hypothermic machine perfusion (HMP) in a porcine model of DCD liver. Female domestic crossbred Large Yorkshire and Landrace pigs weighing approximately 20 kg were used. The abdominal aorta and inferior vena cava were cannulated and connected to an ECMO circuit for in situ perfusion of the abdominal organs at 22 °C for 60 min, 45 min after cardiac death. The pigs were divided into the cold storage (CS) group (n = 3), where liver grafts were preserved at 4 °C, and the HMP group (n = 3), where liver grafts were preserved by HMP at 8-10 °C. After 4 h of preservation, liver function was evaluated using an isolated liver reperfusion model for 2 h. Although the difference was insignificant, the liver effluent enzyme levels in the HMP group were lower than those in the CS group. Furthermore, morphological findings showed fewer injured hepatocytes in the HMP group than in the CS group. The combined use of in situ subnormothermic ECMO and HMP was beneficial for the functional improvement of DCD liver grafts.

Hypothermic Machine Perfusion with Hydrogen Gas Reduces Focal Injury in Rat Livers but Fails to Restore Organ Function.

BACKGROUND: We have previously reported the efficacy of post-reperfusion H2 gas treatment in cold storage (CS) and subsequent reperfusion of the rat liver. The present study aimed to evaluate the effect of H2 gas treatment during hypothermic machine perfusion (HMP) in rat livers retrieved from donation after circulatory death (DCD) and elucidate the mechanism of action of H2 gas. METHODS: Liver grafts were procured from rats after 30 min of cardiopulmonary arrest. The graft was subjected to HMP for 3 hours at 7°C using Belzer MPS with or without dissolved H2 gas. The graft was reperfused using an isolated perfused rat liver apparatus at 37°C for 90 minutes. Perfusion kinetics, liver damage, function, apoptosis, and ultrastructure were evaluated. RESULTS: Portal venous resistance, bile production, and oxygen consumption rates were identical in the CS, MP, and MP-H2 groups. Liver enzyme leakage was suppressed by MP (vs control), whereas H2 treatment did not show a combination effect. Histopathology revealed poorly stained areas with a structural deformity just below the liver surface in the CS and MP groups, whereas these findings disappeared in the MP-H2 group. The apoptotic index in the CS and MP groups was high but decreased in the MP-H2 group. Mitochondrial cristae were damaged in the CS group but preserved in the MP and MP-H2 groups. CONCLUSIONS: In conclusion, HMP and H2 gas treatment are partly effective in DCD rat livers but insufficient. Hypothermic machine perfusion can improve focal microcirculation and preserve mitochondrial ultrastructure.

Ultrastructural changes in porcine liver sinusoidal endothelial cells of machine perfused liver donated after cardiac death.

BACKGROUND: The machine perfusion (MP) preservation including hypothermic MP (HMP) and midthermic MP (MMP) has been considered as a promising strategy to preserve the functions of liver donated after cardiac death. The importance of understanding liver sinusoidal endothelial cells (LSEC) damage in regulating liver injury during MP has been emphasized. However, the ultrastructural changes in the LSEC and sinusoids around them after MP are unclear. AIM: To investigate the ultrastructural changes in the LSEC and sinusoids around them after MP. METHODS: Porcine liver grafts undergo a warm ischemia time of 60 minutes perfused for 4 h with modified University of Wisconsin gluconate solution. Group A grafts were preserved with HMP at 8 °C constantly for 4 h. Group B grafts were preserved with a rewarming solution at 22 °C by MMP for 4 h. Then the ultrastructural changes in the LSEC and sinusoids in Group A and B were comparatively analyzed by using osmium-maceration scanning electron microscopy with complementary transmission electron microscopy methods. RESULTS: An analysis of the LSEC after warm ischemia revealed that mitochondria with condensed-shaped cristae, abnormal vesicles, reduction of ribosomes and the endoplasmic reticulum (ER) surround the mitochondria appeared. The MP subsequent after warm ischemia alleviate the abnormal vesicles and reduction of ribosomes in LSEC, which indicated the reduction of the ER damage. However, MMP could restore the tubular mitochondrial cristae, while after HMP the condensed and narrow mitochondrial cristae remained. In addition, the volume of the sinusoidal space in the liver grafts after MMP were restored, which indicated a lower risk of pressure injury than HMP. CONCLUSION: MMP alleviates the ER damage of LSEC by warm ischemia, additionally restore the metabolism of LSEC via the normalization of mitochondria and prevent the share stress damage of liver grafts.

Identification of potent anti-Cryptosporidium new drug leads by screening traditional Chinese medicines.

Cryptosporidium spp. are gastrointestinal opportunistic protozoan parasites that infect humans, domestic animals, and wild animals all over the world. Cryptosporidiosis is the second leading infectious diarrheal disease in infants less than 5 years old. Cryptosporidiosis is a common zoonotic disease associated with diarrhea in infants and immunocompromised individuals. Consequently, cryptosporidiosis is considered a serious economic, veterinary, and medical concern. The treatment options for cryptosporidiosis are limited. To address this problem, we screened a natural product library containing 87 compounds of Traditional Chinese Medicines for anti-Cryptosporidium compounds that could serve as novel drug leads and therapeutic targets against C. parvum. To examine the anti-Cryptosporidium activity and half-maximal inhibitory doses (EC50) of these compounds, we performed in vitro assays (Cryptosporidium growth inhibition assay and host cell viability assay) and in vivo experiments in mice. In these assays, the C. parvum HNJ-1 strain was used. Four of the 87 compounds (alisol-A, alisol-B, atropine sulfate, and bufotalin) showed strong anti-Cryptosporidium activity in vitro (EC50 values = 122.9±6.7, 79.58±13.8, 253.5±30.3, and 63.43±18.7 nM, respectively), and minimum host cell cytotoxicity (cell survival > 95%). Furthermore, atropine sulfate (200 mg/kg) and bufotalin (0.1 mg/kg) also showed in vivo inhibitory effects. Our findings demonstrate that atropine sulfate and bufotalin are effective against C. parvum infection both in vitro and in vivo. These compounds may, therefore, represent promising novel anti-Cryptosporidium drug leads for future medications against cryptosporidiosis.

Seasonal ultrastructural changes in apocrine gland cells in back skin of male brown bears (Ursus arctos).

Oily secretions from the back skin are involved in the marking behavior of male brown bears (Ursus arctos), and apocrine glands in back skin are activated during the breeding season. Here, we investigated seasonal changes in the intracellular organelles of apocrine gland cells in the back skin of male brown bears using transmission electron microscopy (TEM) and osmium-maceration scanning electron microscopy (OM-SEM). The morphological features of mitochondria and intracellular granules, and secretory mechanisms obviously differed between breeding and non-breeding seasons. The TEM findings showed that contents of low-density granules were released into the glandular lumen by frequent exocytosis, and sausage-shaped mitochondria were located in the perinuclear region during the non-breeding season. In contrast, high-density granules appeared in the apical region and in projections during the breeding season, and swollen mitochondria and lysosome-like organelles separating into high-density granules were located in the perinuclear region. The OM-SEM findings revealed swollen mitochondria with only a few partially developed cristae, and small mitochondria with cristae shaped like those in swollen mitochondria in the apical regions during the breeding season. These findings indicated that the small mitochondria corresponded to the high-density granules identified by TEM. These findings suggested that mitochondria in apocrine gland cells swell, degenerate, fracture into small pieces, and are finally released by apocrine secretions during the breeding season. Small mitochondria released in this secretory manner might function as the source of chemical signals in the oily secretions of brown bears during the breeding season.

Histological findings of sperm storage in green turtle (Chelonia mydas) oviduct.

Green turtles (Chelonia mydas) are seasonal breeders with a time lag between mating and nesting periods. We therefore investigated whether female turtles store sperm like some other animals by histologically and ultrastructurally analyzing oviducts collected from three mature female free-ranging green turtles during the breeding season in the Ogasawara Islands, Japan. The oviduct comprised an infundibulum, magnum, isthmus, uterus, and vagina. Sperm was found in the isthmus of all turtles examined. Some spermatozoa were found in the duct and acini of glands in the isthmus of two turtles with oviducts containing eggs, and a few were also located in the transition area between the uterus and vagina of one of the turtles. On the other hand, we also found abundant spermatozoa on the luminal surface of the isthmus of one turtle captured during mating. In most reptiles, fertilization occurs in the infundibulum or albumen region, and thus the isthmus near those areas might be suitable for storing sperm in female turtles.

Thiamine treatment preserves cardiac function against ischemia injury via maintaining mitochondrial size and ATP levels.

Thiamine (vitamin B1) is necessary for energy production, especially in the heart. Recent studies have demonstrated that thiamine supplementation for cardiac diseases is beneficial. However, the detailed mechanisms underlying thiamine-preserved cardiac function have not been elucidated. To this end, we conducted a functional analysis, metabolome analysis, and electron microscopic analysis to unveil the mechanisms of preserved cardiac function through supplementation with thiamine for ischemic cardiac disease. Male Sprague-Dawley rats (around 10 wk old) were used. Following pretreatment with or without thiamine pyrophosphate (TPP; 300 µM), hearts were exposed to ischemia (40 min of global ischemia followed by 60 min of reperfusion). We measured the left ventricle developed pressure (LVDP) throughout the protocol. The LVDP during reperfusion in the TPP-treated heart was significantly higher than that in the untreated heart. Metabolome analysis was performed using capillary electrophoresis-time-of-flight mass spectrometry, and it revealed that the TPP-treated heart retained higher adenosine triphosphate (ATP) levels compared with the untreated heart after ischemia. The metabolic pathway showed that there was a significant increase in fumaric acid and malic acid from the tricarboxylic acid cycle following ischemia. Electron microscope analysis revealed that the mitochondria size in the TPP-treated heart was larger than that in the untreated heart. Mitochondrial fission in the TPP-treated heart was also inhibited, which was confirmed by a decrease in the phosphorylation level of DRP1 (fission related protein). TPP treatment for cardiac ischemia preserved ATP levels probably as a result of maintaining larger mitochondria by inhibiting fission, thereby allowing the TPP-treated heart to preserve contractility performance during reperfusion.NEW & NOTEWORTHY We found that treatment with thiamine can have a protective effect on myocardial ischemia. Thiamine likely mediates mitochondrial fission through the inhibition of DRP1 phosphorylation and the preservation of larger-sized mitochondria and ATP concentration, leading to higher cardiac contractility performance during the subsequent reperfusion state.

The ultrastructural characteristics of bile canaliculus in porcine liver donated after cardiac death and machine perfusion preservation.

The effects of each type of machine perfusion preservation (MP) of liver grafts donated after cardiac death on the bile canaliculi of hepatocytes remain unclear. We analyzed the intracellular three-dimensional ultrastructure of the bile canaliculi and hepatocyte endomembrane systems in porcine liver grafts after warm ischemia followed by successive MP with modified University of Wisconsin gluconate solution. Transmission and osmium-maceration scanning electron microscopy revealed that lumen volume of the bile canaliculi decreased after warm ischemia. In liver grafts preserved by hypothermic MP condition, bile canaliculi tended to recover in terms of lumen volume, while their microvilli regressed. In contrast, midthermic MP condition preserved the functional form of the microvilli of the bile canaliculi. Machine perfusion preservation potentially restored the bile canaliculus lumen and alleviated the cessation of cellular endocrine processes due to warm ischemia. In addition, midthermic MP condition prevented the retraction of the microvilli of bile canaliculi, suggesting further mitigation of the damage of the bile canaliculi.