Language：English   Publishing type：Research paper (scientific journal)   Publisher：Adipocyte  

Normalization is a crucial step in gene expression analysis to avoid misinterpretation. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of 10 candidate housekeeping genes in non-differentiated (ND) and differentiated (DI) 3T3-L1 cells on days 5 and 10. We used geNorm, NormFinder, BestKeeper, RefFinder, and the ∆Ct method to evaluate expression stability. The findings revealed that (1) the expression levels of the reference genes changed over time, even in non-differentiating cells, and (2) peptidylprolyl isomerase A (Ppia) and TATA box-binding protein (Tbp) were stable reference genes for 10 days in both undifferentiated and differentiated 3T3-L1 cells. Notably, the expression of known reference genes in non-differentiating cells was altered throughout the experiment.

DOI： 10.1080/21623945.2023.2235081

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Animal Science Journal  

Chondroitin sulfate/dermatan sulfate (CS/DS) is a member of glycosaminoglycans (GAGs) found in animal tissues. Major CS/DS subclasses, O, A, C, D, and E units, exist based on the sulfation pattern in d-glucuronic acid (GlcA) and N-acetyl-d-galactosamine repeating units. DS is formed when GlcA is epimerized into l-iduronic acid. Our study aimed to analyze the CS/DS profile in 3 T3-L1 cells before and after adipogenic induction. CS/DS contents, molecular weight (Mw), and sulfation pattern were analyzed by using high-performance liquid chromatography. CS/DS synthesis- and sulfotransferase-related genes were analyzed by reverse transcription real-time PCR. CS/DS amount was significantly decreased in the differentiated (DI) group compared to the non-differentiated (ND) group, along with a lower expression of CS biosynthesis-related genes, chondroitin sulfate N-acetylgalactosaminyltransferase 1 and 2, as well as chondroitin polymerizing factor. GAGs in the DI group also showed lower Mw than those of ND. Furthermore, the A unit was the major CS/DS in both groups, with a proportionally higher CS-A in the DI group. This was consistent with the expression of carbohydrate sulfotransferase 12 that encodes chondroitin 4-O-sulfotransferase, for CS-A formation. These qualitative and quantitative changes in CS/DS and CS/DS-synthases before and after adipocyte differentiation reveal valuable insights into adipocyte development.

DOI： 10.1111/asj.13894

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Poultry Science Association  

<p>Collagen content and collagen fiber architecture in the skin of Shamo chickens were compared between sexes and body parts. Cervical, thoracic, dorsal, femoral, and crural skin samples were collected and their collagen content was analyzed. Collagen fiber specimens were prepared for scanning electron microscopy using the cell maceration method with a NaOH solution. Sex differences in collagen content were only observed in the femoral skin of mature chickens, but not in 10-week-old chicks. The difference in collagen content between body parts was obvious; femoral and crural skin had higher collagen content than those of other parts in both sexes. Scanning electron microscopy indicated that the collagen fiber architecture was quite different between the superficial and deep layers in the dermis, with the former consisting of loosely tangled band-like collagen fibers, and the latter composed of thick and dense layers of collagen bundles in a parallel arrangement. The width of collagen fibers in the superficial layer of the dermis differed between sexes in the dorsal, femoral, and crural skin. From these results, it is likely that the difference in collagen content in the femoral skin is not due to sex hormones but other factors, such as mechanical stimulation in daily activity. Additionally, collagen fiber width in the superficial layer is likely related to the difference in collagen content between sexes and between body parts.</p>

DOI： 10.2141/jpsa.2023026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Animal Science Journal  

Chicken adenohypophyseal cells were cultured in plates coated with different materials, and their morphologies were examined to confirm the characteristics of chicken folliculo-stellate (FS) cells in vitro. The adenohypophyseal cells were dispersed with a collagenase/trypsin mixture in media and seeded in plates coated in either poly L-lysine (PLL), collagen, or laminin. After 7 days of culture, the cells were fixed and immunocytochemistry was performed. 5-Bromo-2'-deoxyuridine incorporation test indicated that the proliferation activity of the culture cells was different based on the coating materials, and it was higher in the collagen-coated plate than two other coating materials. Fluorescence immunocytochemistry was also performed using mixed antibodies against growth hormone, prolactin, luteinizing hormone β-subunit, basic cytokeratin (bCK), and S100B. The culture cells on the PLL- and laminin-coated surfaces were round or oval in shape, and bCK-immunopositive FS cells were morphologically indistinguishable from endocrine cells. In the collagen-coated plate, many endocrine cells were round or oval in shape, but FS cells displayed a larger and flattened morphology. S100B-immunoreactions were localized in the nuclei of bCK-immunopositive FS cells. These results suggest that culturing the chicken adenohypophyseal cells in the collagen-coated plate enables the distinction of FS cells from endocrine cells.

DOI： 10.1111/asj.13814

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Veterinary Medicine Series C: Anatomia Histologia Embryologia  

The complexity of the Sunda porcupine skin has become an important topic due to the unique characteristics of its quill follicles. The structure and chemical composition of the skin has affected many physiological and other conditions. Generally, quills are larger, stronger and stiffer than hair; therefore, the skin structure needs to adapt to support their physiology. The strength of the skin is determined by its collagen composition and arrangement; therefore, this study aims to analyse the composition and distribution of thick and thin fibres based on the specific characteristics of Sunda porcupine skin under polarized light using picrosirius red staining. The skin samples used were from the thoracodorsal and lumbosacral regions of eight Sunda porcupine adults. The histological staining was carried out using the picrosirius red method, while the samples were observed under a polarized light microscope and analysed with software. The results showed that the skin is composed of 36%-65% thick fibres, 20%-35% thin fibres and small amounts of other types with the lumbosacral region having higher compositions of thick and thin fibres than those in the thoracodorsal region. Furthermore, the thoracodorsal and lumbosacral regions have the highest composition of thick fibre in the deeper dermis and quill follicle, respectively. These demonstrated that the complexity of the skin structure of Sunda porcupine due to its quill derivates correlated with its collagen composition and distribution.

DOI： 10.1111/ahe.12845

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

Glycosaminoglycans (GAGs) are found in the various tissues and are involved in many physiological functions. Since the rhesus monkey (Macaca mulatta) is the most widely used nonhuman primate in biomedical research, an understanding of the compositions of GAGs in their tissues is important. The aim of the study was to determine the content and sulfation pattern of disaccharides contained in several tissues of the rhesus monkey. The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from several tissues of female and male rhesus monkeys. Compositional analysis was performed after digestion with chondroitinases ABC and ACI to reveal the sulfation pattern of the CS/DS hybrid chain. This study revealed that the major CS/DS disaccharide units present in the tissues were A- and C-types. The E- and iE-types were specifically distributed not only in the tracheal tissue but also in gastrointestinal tissues.

DOI： 10.1093/bbb/zbac057

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Veterinary Medical Science  

Myogenesis, the formation of muscle fibers, is affected by certain glycoproteins, including chondroitin sulfate (CS), which are involved in various cellular processes. We aimed to investigate the mechanism underlying CS-E-induced suppression of myotube formation using the myoblast cell line C2C12. Differentiated cells treated with 0.1 mg/ml CS-E for nine days showed multinucleated and rounded myotubes with myosin heavy chain positivity. No difference was found between the CS-E-treated group with rounded myotubes and CS (-) controls with elongated myotubes in the levels of phospho-cofilin, a protein involved in the dynamics of actin cytoskeleton. Interestingly, N-cadherin, which is involved in the gene expression of myoblast fusion factors (myomaker and myomixer), was significantly downregulated at both the mRNA and protein levels following CS-E treatment. These results suggest that N-cadherin downregulation is one of the mechanisms underlying the CS-E-induced suppression of myotube formation.

DOI： 10.1292/jvms.21-0662

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Veterinary Medical Science  

In this study, we induced chemical damage of C2C12 myoblasts that had differentiated into myotubes with glycerol, and four sulfation enzymes for chondroitin sulfate (CS) [carbohydrate sulfotransferase (Chst)12, Chst15 and Chst3) and uronyl 2-O-sulfotransferase (UST)] and two CS degradation enzymes [hyaluronidase (Hyal)1 and Hyal2] were examined for changes in gene expression. Treatment of myoblasts with 5% glycerol significantly increased the expression levels of the sulfation enzymes Chst12 and Chst15 and the degradation enzymes Hyal1 and Hyal2. However, the expression levels of the other two genes (Chst3 and Ust) showed no change. Differences in the expression levels of these enzymes may help to understand the difference in responsiveness of myoblasts to glycerol after muscle injury in vivo or in vitro.

DOI： 10.1292/jvms.21-0632

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The purpose of this study was to elucidate the functions of estrogen and two estrogen receptors (ERs; ERα and ERβ) in the myoregeneration process and morphogenesis. Cardiotoxin (CTX) was injected into the tibialis anterior (TA) muscles of ovariectomized (OVX) mice to induce muscle injury, and subsequent myoregeneration was morphologically assessed. The diameter of regenerated myotubes in OVX mice was significantly smaller than that in intact mice at all time points of measurement. OVX mice also showed lower muscle recovery rates and slower speeds than did intact mice. ER protein levels showed a predominance of ERβ over ERα in both intact and OVX states. The ERβ level was increased significantly at 7 days after CTX injection in OVX mice and remained at a high level until 14 days. In addition, continuous administration of E2 to OVX mice in which muscle injury was induced resulted in a significantly larger diameter of regenerated myotubes than that in mice that did not receive estrogen. The results indicate that estrogen is an essential factor in the myoregeneration process since estrogen depletion delayed myoregeneration in injured muscles and administration of estrogen under the condition of a low estrogen status rescued delayed myoregeneration. The results strongly suggested that ERβ may be a factor that promotes myoregeneration more than does ERα.

DOI： 10.1292/jvms.21-0495

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The activation of α2 adrenergic receptors contributes to analgesia not only in the central nervous system but also in the peripheral nervous system. We reported that noradrenaline inhibits the activity of transient receptor potential vanilloid 1 (TRPV1) evoked by capsaicin through α2 receptors in cultured rat dorsal root ganglion (DRG) neurons. However, it is unclear whether activation of TRPV1 expressed in peripheral nerve terminals is inhibited by α2 receptors and whether this phenomenon contributes to analgesia. Therefore, we examined effects of clonidine, an α2 receptor agonist, on several types of nociceptive behaviors, which may be caused by TRPV1 activity, and subtypes of α2 receptors expressed with TRPV1 in primary sensory neurons in rats. Capsaicin injected into hind paws evoked nociceptive behaviors and clonidine preinjected into the same site inhibited capsaicin-evoked responses. This inhibition was not observed when clonidine was injected into the contralateral hind paws. Preinjection of clonidine into the plantar surface of ipsilateral, but not contralateral, hind paws reduced the sensitivity to heat stimuli. Clonidine partially reduced formalin-evoked responses when it was preinjected into ipsilateral hind paws. The expression level of α2C receptor mRNA quantified by real-time PCR was highest followed by those of α2A and α2B receptors in DRGs. α2A andα2C receptor-like immunoreactivities were detected with TRPV1-like immunoreactivities in the same neurons. These results suggest that TRPV1 and α2 receptors are coexpressed in peripheral nerve terminals and that the functional association between these two molecules causes analgesia.

DOI： 10.1292/jvms.21-0429

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Epithelial-mesenchymal transition (EMT) in primary tumor cells is a key prerequisite for metastasis initiation. Statins, cholesterol-lowering drugs, can delay metastasis formation in vivo and attenuate the growth and proliferation of tumor cells in vitro. The latter effect is stronger in tumor cells with a mesenchymal-like phenotype than in those with an epithelial one. However, the effect of statins on epithelial cancer cells treated with EMT-inducing growth factors such as transforming growth factor-β (TGF-β) remains unclear. Here, we examined the effect of atorvastatin on two epithelial cancer cell lines following TGF-β treatment. Atorvastatin-induced growth inhibition was stronger in TGF-β-treated cells than in cells not thusly treated. Moreover, treatment of cells with atorvastatin prior to TGF-β treatment enhanced this effect, which was further potentiated by the simultaneous reduction in the expression of the statin target enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Dual pharmacological targeting of HMGCR can thus strongly inhibit the growth and proliferation of epithelial cancer cells treated with TGF-β and may also improve statin therapy-mediated attenuation of metastasis formation in vivo.

DOI： 10.1038/s41598-021-91928-3

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This study aimed to investigate the function of estrogen receptors (ERs) in myoregeneration and intermuscular adipogenesis. Ovariectomized (OVX) ERα knockout (KO) mice and ERβ KO mice were used to assess the effect of estrogen on the myoregenerative process. Tibialis anterior muscle was collected on days 7, 10, and 14 after cardiotoxin injection to assess myotube morphology and adipogenesis area. Regenerated myotubes from OVX-ERβ KO mice were consistently smaller in diameter than those from OVX-ERα KO and OVX-wild-type mice, whereas the adipogenesis area of OVX-ERβ KO mice was consistently greater than that of the other types. Therefore, ERβ may be an influential factor in promoting myoregeneration and adipogenesis inhibition compared to ERα.

DOI： 10.1292/jvms.20-0696

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Uncoupling protein 1 (UCP-1) was believed to be an exclusive protein found in the brown adipose tissue of small rodents and humans; however, recent studies show that the expression of UCP-1 protein has been found in the sebaceous glands of the mouse tail and human skin. There are a few reports about the presence of UCP-1 in the sebaceous glands of other rodents, such as the Sunda porcupine (Hystrix javanica), a wild spiny rodent commonly found in Indonesia with a large sebaceous gland. The aim of this study was to identify the presence of UCP-1 in the sebaceous glands on the skin of the Sunda porcupine. The skin from three regions (thoracodorsal, lumbosacral and apex caudal) of eight adult Sunda porcupines was used to detect UCP-1-immunopositive cells through immunohistochemistry. All three regions were found immunopositive to anti-UCP-1 antibody in the sebaceous gland of quill and hair follicles, and the epidermal layer in quill and hair follicles with various intensities. The result of immunohistochemistry revealed that the thoracodorsal and apex caudal region was the most intense immunoreaction followed by the lumbosacral region. These findings proved that the presence of UCP-1 was also identified in the sebaceous glands of other rodent (Hystrix javanica) and regions of the body, which has not been reported previously.

DOI： 10.1292/jvms.20-0412

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This study aimed to investigate the metabolic effect of yogurt fermented by Lactobacillus fermentum TSI and S2 isolated from a Mongolian traditional dairy product on rats with high-fat-diet-induced obesity. Quality characteristics of yogurt fermented by commercial starter (CON), L. fermentum TSI2 (TSI2 group), L. fermentum S2 (S2 group), and mixed TSI2 and S2 strains at 1:1 (MIX group), were verified. Six-week-old male Sprague-Dawley rats were divided into five groups and administered the following diets: group NOR, normal diet with oral saline administration; group HF, high-fat diet (HD) with oral saline administration; group TSI, HD and L. fermentum TSI-fermented yogurt; group S2, HD and L. fermentum S2-fermented yogurt; and group MIX, HD and MIX-fermented yogurt. After eight weeks, the HD groups displayed significantly increased body weight and fat, serum cholesterol, and abdominal adipose tissue levels. However, serum HDL cholesterol levels were higher, triglyceride levels were lower, and abdominal adipocytes were smaller in the TSI and S2 groups than in the HF group. These results indicate that L. fermentum TSI reduces abdominal fat and improves blood lipid metabolism in HD-induced obese rats.

DOI： 10.3390/foods9050594

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Wound healing in the Sunda porcupine is believed to occur quickly, although the wound is large and severe. Wound enclosure involves many processes to restore the lost or damaged skin structure where conjugated polysaccharide-protein and collagen, as the main components deposited in wound tissue to restore it. The aim of this study was to evaluate alteration of polysaccharide contents and collagen in untreated full-thickness wound healing in the thoracodorsal and lumbosacral regions in the Sunda porcupines. Histological analysis was performed by periodic acid Schiff, alcian blue pH 2.5, picrosirius red staining method and Low Vacuum Scanning Electron Microscope (LV-SEM) imaging to obtain the fundamental data of healing process. Wound healing began with re-epithelization followed by progressive wound contraction with 4 overlapping stages in about 30-50 days until the wound closed (21-30 days in thoracodorsal and 30-50 days in lumbosacral). Neutral polysaccharide was more widely distributed compared to the acid polysaccharide in almost all stages of wound healing. The ratio of collagen I to III appeared to be higher in the thoracodorsal region than the lumbosacral region during healing process. LV-SEM imaging showed changes in connective tissue structure in the wound border and granulation tissue which appeared abundant and mixed of thin and thick fiber. In conclusion, cutaneous full thickness wound healing in the Sunda porcupine occurred faster in the thoracodorsal region, which might be correlated to the role of neutral polysaccharide and a high ratio of collagen I to III.

DOI： 10.1292/jvms.19-0603

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Neutralization of transforming growth factor (TGF)-β1 activity reduced fibrosis and enhanced regeneration of glycerol-injured rat muscle.
Recently, we have shown that glycerol induces early fibrosis in rat muscles which persists up to two weeks after injury. The current study aims to determine the possible factor associated with fibrosis of rat muscle following glycerol injury. Eight-week-old male Wistar rats received either glycerol only (as a control) or a co-treatment of neutralizing antibody to transforming growth factor (TGF)-β1 (5 and 12.5 µg). Both antibody doses significantly decreased fibrosis and improved muscle regeneration suggesting that anti-TGF-β1 antibody has both anti-fibrotic and myogenic effects. In conclusion, fibrosis developed in glycerol-injured rat muscles, might be mediated, in part, by the upregulation of TGF-β1 expression. Targeting TGF-β1 could be a promising approach for inhibiting fibrosis and enhancing muscle regeneration.

DOI： 10.1292/jvms.19-0446

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Glycosaminoglycans (GAG) from the velvet antlers of Sika deer (Cervus nippon) at the different growing stages (Fukurozuno, Anshi, and Santajo) of bred and wild deer were isolated and their concentrations and sulfation patterns were analyzed. GAG were digested with chondroitinase ABC, ACI, heparinase-I and -III, and keratanase-II into the corresponding repeating disaccharides of chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan, heparan sulfate (HS), and keratan sulfate. Cartilaginous tissues contained CS-DS at high concentrations with an almost equal ratio of 4- and 6-sulfates, while 4-sulfate-type CS-DS predominantly occupied ossified tissues, but at low concentrations. High O- and N-sulfation degrees of HS correspond to high ossification. Dynamic quantitative changes in CS-DS and compositional changes in CS-DS and HS were closely associated with the mineralization of deer antlers.

DOI： 10.1007/s10719-019-09859-1

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This study evaluated the expression of genes involved in the concentration of Ca2+ in precursor osteoblast-like cell, MC3T3-E1 subjected to stretching stimuli. Transient receptor potential vanilloid 4 (Trpv4) gene expression, the factor that is activated by stretch stimulation and enables inflow of Ca2+ from the extracellular space, was not affected as a result of stretch stimulation; conversely, the expression of sodium-calcium exchanger 1 (Ncx1) gene involved in outflow of intracellular Ca2+ increased, depending on stimulation intensity. Localization of Ca2+ correlated with the positioning of the endoplasmic reticulum, and intracellular Ca2+ decreased in inverse proportion to the intensity of the stretching force. These results suggest that stretch stimulation activates intracellular Ca2+ elimination rather than Ca2+ uptake before osteoblast differentiation.

DOI： 10.1292/jvms.18-0766

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Glycerol has been recently used to induce muscle adiposity in mice. However, its effects on the rat muscles have not been investigated previously. Therefore, we investigated the regeneration outcomes of rat muscles following glycerol-induced injury at different time points. Glycerol injection induced myofiber degeneration with extensive inflammatory infiltration on day 4 followed by appearance of regenerating myotubes on day 7 after injury without adipocyte infiltration. Meanwhile, a significant collagen deposition at early stage of regeneration that increased together with persistent inflammatory infiltration up to day 14 after injury indicates impaired regeneration. In conclusion, glycerol injury in rats is more suitable as a fibrosis-inducing model than in mice due to earlier and higher accumulation of fibrous tissue with lacking adipogenesis.

DOI： 10.1292/jvms.18-0328

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HMG-CoA reductase (HMGCR) inhibitors, statins, are potent cholesterol reducing drugs that exhibit anti-tumor effects in vitro and in animal models, including attenuation of metastasis formation, and their use correlates with reduced cancer-specific mortality in retrospective human cohort studies. However, E-cadherin expressing epithelial- and mixed epithelial-mesenchymal cancer cell lines (reflective of primary and outgrowing metastatic tumor cells, respectively) require higher statin concentrations than mesenchymal-like tumor cells (reflective of in-circulation metastatic tumor cells) to achieve the same degree of growth inhibition. Here, we show that attenuation of HMGCR expression in the presence of atorvastatin leads to stronger growth inhibition than dual target blockade of the mevalonate pathway in relatively statin resistant cell lines, mainly through inhibition of protein prenylation pathways. Thus, combined inhibition of the mevalonate pathway's rate-limiting enzyme, HMGCR, can improve atorvastatin's growth inhibitory effect on epithelial- and mixed mesenchymal-epithelial cancer cells, a finding that may have implications for the design of future anti-metastatic cancer therapies.

DOI： 10.18632/oncotarget.25448

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Osteoblast activating peptide (OBAP) is a newly discovered peptide detected in the rat stomach, which has a major role in osteogenesis. Recently, we revealed its localization in the parietal cells of the rat stomach. There have been no data regarding OBAP expression in the kidney, despite its role in calcium reabsorption in renal tubules. The current study aimed to inspect the expression of OBAP in the kidney of twelve 10-week-old male C3H/HeNJc1 mice using immunohistochemistry, and immunoelectron microscopic localization. The immunohistochemical investigation revealed an OBAP positive reaction mainly in the medulla, which was stronger than the cortex of the kidney and was concentrated in the distal convoluted tubules (DCT), connecting tubules (CT), and the thick limbs of the loop of Henle (HL). Moreover, we clarified that the OBAP was co-distributed with ghrelin and calbindin (markers of the DCT). Interestingly, immunoelectron microscopy demonstrated that OBAP was concentrated in the mitochondrial inner membrane of the DCT and CT. Based on these results, it was concluded that the mitochondria of the DCT, CT, and HL of the mice kidney generate OBAP. Furthermore, our results suggest that OBAP might have a role in the regulation of calcium reabsorption by the renal tubule; however, further investigations are required to clarify this potential role.

DOI： 10.1016/j.acthis.2018.03.001

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Temperature-sensitive gels (TSGs) are generally used in the fields of medical, robotics, MEMS, and also in daily life. In this paper, we synthesized a novel TSG with good thermal durability and a lower melting temperature below 60 °C. We discussed the physical properties of he TSG and found it provided excellent thermal expansion. Therefore, we proposed the usage of TSG to develop a strategic breathable film with controllable gas permeability. The TSG particles were prepared firstly and then blended with linear low-density polyethylene/calcium carbonate (LLDPE/CaCO₃) composite to develop microporous TSG/LLDPE/CaCO₃ films. We investigated the morphology, thermal, and mechanical properties of TSG/LLDPE/CaCO₃ composite films. The film characterization was conducted by gas permeability testing and demonstration temperature control experiments. The uniformly porous structure and the pore size in the range of 5⁻40 μm for the TSG/LLDPE/CaCO₃ composite films were indicated by SEM micrographs. The demonstration temperature control experiments clearly proved the effect of the controllable gas permeability of the TSG and, more promisingly, the great practical value and application prospects of this strategic effect for the temperature-sensitive breathable film was proved.

DOI： 10.3390/polym10050486

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Effects of transforming growth factor-β1 treatment on muscle regeneration and adipogenesis in glycerol-injured muscle
Transforming growth factor (TGF)-β1 is associated with fibrosis in many organs. Recent studies demonstrated that delivery of TGF-β1 into chemically injured muscle enhances fibrosis. In this study, we investigated the effects of exogenous TGF-β1 on muscle regeneration and adipogenesis in glycerol-injured muscle of normal mice. Tibialis anterior (TA) muscles were injured by glycerol injection. TGF-β1 was either co-injected with glycerol, as an 'early treatment' group, or injected at day 4 after glycerol, as a 'late treatment' group and the TA muscles were collected at day 7 after initial injury. Myotube density was significantly lower in the early treatment group than in the glycerol-injured group (without TGF-β1 treatment). Moreover, the Oil red O-positive area was significantly smaller in the early treatment group than in the late treatment group and glycerol-injured group. Furthermore, TGF-β1 treatment increased endomysial fibrosis and induced immunostaining of α-smooth muscle actin. The greater inhibitory effects of early TGF-β1 treatment than that of late TGF-β1 treatment during regeneration in glycerol-injured muscle suggest a more potent effect of TGF-β1 on the initial stage of muscle regeneration and adipogenesis. Combination of TGF-β1 with glycerol might be an alternative to enhance muscle fibrosis for future studies.

DOI： 10.1111/asj.12845

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In this study, we investigated the early changes of skeletal muscle damage in response to injuries induced by cardiotoxin (CTX) and glycerol by using both light microscopy and transmission electron microscopy. Normal, non-dystrophic, adult male mice were used in this study. Tibialis anterior (TA) muscles were injected either with CTX or glycerol. Samples were collected at intervals starting from 1h up to 4days after injury. Injured muscles were subjected to both histological and ultrastructural analyses. CTX-induced injury caused mitochondrial accumulation and swelling followed by lysis, while glycerol-induced injury caused accumulation of vesicles with focal disruption of the basal lamina, indicating that the injuries have different mechanisms of damage to myofibers. Moreover, inflammatory cells, including neutrophils and macrophages, were recruited earlier and in larger numbers after CTX-induced injury than after glycerol-induced injury. On the other hand, satellite cells (SCs) activation started at 6h after both injuries, as indicated by an increase in both the length and cytoplasmic-to-nuclear ratio. However, there were significantly longer SCs with a higher cytoplasmic-to-nuclear ratio in the CTX-injured muscles than in the glycerol-injured muscles at day 4. In conclusion, our results demonstrated a difference between CTX and glycerol in their damage to myofibers; CTX damages myofiber mitochondria, while glycerol damages the myofiber cell membrane and alters osmosis. In addition, CTX-induced injury caused earlier and more extensive inflammatory infiltration than did glycerol-induced injury. This study is the first study to shed light on the early events following skeletal muscle injury induced by CTX and glycerol.

DOI： 10.1016/j.micron.2016.09.009

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Degree of Suppression of Mouse Myoblast Cell Line C₂C12 Differentiation Varies According to Chondroitin Sulfate Subtype.
Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C₂C12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion.

DOI： 10.3390/md14100193

Language：English   Publishing type：Research paper (scientific journal)  

Osteoblast activating peptide (OBAP) was previously reported to be expressed in the rat stomach and to have a vital role in osteogenesis, but its distribution in rat stomach has not been determined. Thus, the aim of the present study was to identify the cell types expressing OBAP in the rat stomach. The stomachs of twelve 10-to-11-week-old male Jc1:SD rats were used. Samples were collected for immunohistochemistry, immunoelectron microscopy and dot blot assay. Immunohistochemical investigation revealed that OBAP was distributed mainly in parietal cells without any expression in chief cells, X/A-like cells or enterochromaffin-like cells. Moreover, OBAP-immunopositive cells were observed mainly in the upper and lower parts of the gastric gland. Significantly high optical density of immunopositive cells was observed in the upper and lower gastric gland regions. The dot blot assay confirmed that OBAP is secreted by parietal cells and that it is present in the gastric gland lumen. Immunoelectron microscopy demonstrated that OBAP was confined to the mitochondrial inner membrane within parietal cells and that the number of mitochondria in the upper and lower parts of the gastric epithelium was significantly larger than the number in the middle part of the gastric epithelium. Based on the results, it was concluded that OBAP is mainly produced by mitochondria of parietal cells in the upper and lower parts of the gastric epithelium. Moreover, the presence of OBAP in the gastric gland lumen suggests an exocrine mechanism of release.

DOI： 10.1016/j.acthis.2015.12.001

Language：English   Publishing type：Research paper (scientific journal)  

Repair of segmental ulna defects using a β-TCP implant in combination with a heparan sulfate glycosaminoglycan variant
© 2015 Acta Materialia Inc. Given the wide spread clinical use of ceramic-based bone void fillers, we sought to determine the efficacy of an FDA-approved β-tricalcium phosphate bone graft substitute (JAX™) in combination with a carboxymethyl cellulose (CMC) handling agent that included a particular heparan glycosaminoglycan (GAG) variant, herein referred to as HS3. Having recently demonstrated efficacy of a combination collagen/HS3 device, we further aimed to determine the support that HS3 could offer a handling agent used to administer a more tissue-relevant bone void filler. This study evaluated the JAX™-HS3 combination device in 1.5 cm critical-sized defects in the ulna bones of 27 male New Zealand White rabbits. Treatment groups consisted of JAX™ applied with CMC alone, or JAX™ with CMC containing either 30 μg or 100 μg of the HS3 GAG. Data based on radiographic, μCT, mechanical, and histological analyses at 4 and 8 weeks post-surgery, clearly demonstrate enhanced new bone formation in the JAX™-HS3 combination treated defects compared to treatment with JAX™ alone. The efficacy of such a combination advocates for inclusion of HS3 in handling agents used in the preparation of various bone void fillers being used in orthopaedic surgery. Statement of significance Synthetic bone grafts and demineralized bone matrices are gaining prominence as alternatives to autologous and allogeneic bone grafts and are frequently administered in granular form, necessitating their combination with a handling agent. Typical handling agents include glycerol, gelatin, cellulose, hyaluronic acid and lecithin, formulated as hydrogels, which can be further enhanced by the addition of heparan sulfate (HS) glycosaminoglycans that augment the osteostimulatory properties of the graft. Here we assessed the efficacy of β-TCP granules combined with a hydrogel consisting of carboxymethyl cellulose and the HS variant (HS3) previously shown to enhance osteogenic healing. The data advocates for HS3 to be included during the formulation of hydrogel-based carriers that support the various bone void fillers being used in orthopaedic surgery.

DOI： 10.1016/j.actbio.2015.09.008

Language：English   Publishing type：Research paper (scientific journal)  

In the present study, we examined muscle regeneration following two types of chemical injuries, cardiotoxin (CTX) and glycerol, in order to compare their effect on the morphological characteristics during muscle regeneration, in addition we studied the structural changes of the intramuscular connective tissue (IMCT) during the regeneration process, by scanning electron microscopy (SEM) after digestion of the cellular elements of the muscle with sodium hydroxide. Tibialis anterior (TA) muscles of adult male mice were injected either with CTX or glycerol. Muscle degeneration was greater in the CTX-injured model than in the glycerol-injured model at day 4 post injection. Muscle regeneration started at day 7 in both the CTX and glycerol models. However, the CTX-injured model showed a higher myotube density and larger myotube diameter than the glycerol-injured model at days 10 and 14 post injection. On other hand, adipocyte infiltration was detected in the glycerol-injured model. In contrast, no adipocytes could be detected in the CTX-injured model. Furthermore, ultrastructural analysis showed a significant difference in myofiber damage and regeneration between the two models. SEM of the IMCT showed a transient increase in endomysial collagen deposition at early stages of regeneration in the CTX-injured model. In contrast, glycerol-injured model showed slight endomysial collagen deposition. Our results suggest that changes in IMCT affect the efficiency of muscle regeneration. Studying the three dimensional structure of IMCT may help clinical therapies to reduce skeletal muscle fibrosis. To our knowledge this is the first time the changes in IMCT following CTX and glycerol injury using SEM-cell maceration technique have been compared.

DOI： 10.1016/j.aanat.2015.07.002

Language：English   Publishing type：Research paper (scientific journal)  

In teleosts, the spinal cord generally extends along the entire vertebral canal. The Tetraodontiformes, in which the spinal cord is greatly reduced in length with a distinct long filum terminale and cauda equina, have been regarded as an aberration. The aims of this study are: 1) to elucidate whether the spinal cord in all tetraodontiform fishes shorten with the filum terminale, and 2) to describe the gross anatomical and histological differences in the spinal cord among all families of the Tetraodontiformes. Representative species from all families of the Tetraodontiformes, and for comparison the carp as a common teleost, were investigated. In the Triacanthodidae, Triacanthidae, and Triodontidae, which are the more ancestral taxa of the Tetraodontiformes, the spinal cord extends through the entire vertebral canal. In the Triacanthidae and Triodontidae, the caudal half or more spinal segments of the spinal cord, however, lack gray matter and consist largely of nerve fibers. In the other tetraodontiform families, the spinal cord is shortened forming a filum terminale with the cauda equina, which is prolonged as far as the last vertebra. The shortened spinal cord is divided into three groups. In the Ostraciidae and Molidae, the spinal cord tapers abruptly at the cranium or first vertebra forming a cord-like filum terminale. In the Monacanthidae, Tetraodontidae, and Diodontidae, it abruptly flattens at the rostral vertebrae forming a flat filum terminale. The spinal cord is relatively longer in the Monacanthidae than that in the other two families. It is suggested by histological features of the flat filum terminale that shortening of the spinal cord in this group progresses in order of the Monacanthidae, Tetraodontidae, and Diodontidae. In the Balistidae and Aracanidae, the cord is relatively long and then gradually decreased in dorso-ventral thickness.

DOI： 10.1002/jmor.20338

Language：English   Publishing type：Research paper (scientific journal)  

The dog visual system is well suited to dim light conditions due to rod-dominated retina and the reflective tapetum. The topographical distributions of rods and thickness of the tapetum of the dog were quantified in retinal whole mounts stained with thionine, and spatial relationships among the tapetum, rod density and visual streak of high ganglion cell density were elucidated. The relationship between the retina and tapetum was analyzed in parasagittal sections stained with thionine or hematoxylin-eosin. The tapetum was thick in its center, and the thickest part consisted of 9 to 12 tapetal cell layers. Rod density ranged from 200,000 to 540,000/mm(2). Maximum rod density was found in the area dorsal to the visual streak, and the density in that area was significantly higher than the rod density in the visual streak and accorded spatially with the thickest part of the tapetum. The horizontal visual streak was found over the horizontal line through the optic disc in the temporal half and extended slightly into the nasal half. The central area of the highest density of ganglion cells was approximately located midway between the nasal and temporal ends of the visual streak. The visual streak was located within the tapetal area, but ventrally to the thick part of the tapetum.

DOI： 10.1292/jvms.14-0447

Language：English   Publishing type：Research paper (scientific journal)  

Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 (Trpv1)-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1, although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca(2+) concentration ([Ca(2+)]i) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca(2+)]i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca(2+)]i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 μM) and the TRPV1 antagonists, capsazepine (10 μM) and BCTC (10 μM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca(2+)]i elevation when the temperature was increased to 36°C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules.

DOI： 10.1016/j.ceca.2014.11.003

Language：English   Publishing type：Research paper (other academic)  

Language：English   Publishing type：Research paper (scientific journal)  

We aimed to document macroscopic variations in the cellular tapetum in the dog, to provide a histologic description of the macroscopic results and to evaluate the correlation between the macroscopic appearance and aging. Fifty three dogs including 5 beagles, 1 Chihuahua and 47 mixed breeds of each gender were used. For a macroscopic study, the fresh tapetal fundi were photographed using digital camera. For a histological study, the glutaraldehyde-formalin fixed eyes were embedded in nitrocellulose and stained with hematoxylin-eosin or thionine. The normal tapetum was triangular with the rounded angles and the smooth contour. The atypical tapetum was smaller and more variable in shape, contour and color than the normal one. In severe cases, the fundus was devoid of the tapetum. The atypical tapetum tended to increase in frequency with aging. Retinal pigment epithelial cells on the normal tapetum were unpigmented. In the eye with the atypical tapetum, regardless of tapetal size and shape, unpigmented retinal pigment epithelial cells showed a similar distribution to that on the normal tapetum, even in a dog without a tapetum. Although there is a congenitally hypoplastic tapetum, the atypical tapetum tends to increase in incidence and severity with aging.

DOI： 10.1292/jvms.14-0132

Language：English   Publishing type：Research paper (scientific journal)  

Studies of ocular development in the dromedary camel (Camelus dromedarius) have not been reported previously. The aim of the present investigation was therefore to document the major landmarks and the time course in the prenatal development of the eye tunics in dromedary camel and its accommodation with the surrounding hard environment of the desert. Serial histological sections of dromedary camel embryos and foetuses were used. Age estimation was made on the basis of gestational size, crown vertebral-rump length (CVRL), which ranged 1.2-110 cm. The eye of the dromedary camel developed in a similar manner to that of the human and domestic animals eyes; the principal differences were in the time of occurrence of certain developmental events, pigmented peripheral cornea near the limbus, a remarkably thickened Descemet's membrane and pigmentation in the corneo-scleral junction, which represent an adaptive modification in relation to a severe environment. © 2013 Blackwell Verlag GmbH.

DOI： 10.1111/ahe.12069

Language：English   Publishing type：Research paper (scientific journal)  

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.

DOI： 10.1007/s00441-014-1836-6

Language：English   Publishing type：Research paper (scientific journal)  

Central projections originated from a single dorsal root ganglion (DRG) were studied in the chicken focusing on the rostrocaudal extension of primary afferents in each lamina by using anterograde labeling by lectin-HRP injection into either the 15th or the 24th DRG. In the injection into the 15th DRG, labeled fibers (LFs) were found in a wide rostrocaudal range of laminas IV (the spinal segment (SS) 1-20) and V (SS 4-18) and in a narrow range of other laminas. In the injection into the 24th DRG, LFs were distributed in a similar rostrocaudal range in all laminas except for laminas VIII and IX. LFs in laminas VIII and IX were restricted in the tracer injected segment. LFs in the lateral funiculus derived from both the enlargements projected into the rostral lamina III in addition to the lower medulla oblongata. There was little overlap in the extent of the primary terminal areas from both the enlargements.

DOI： 10.1016/j.neulet.2013.12.052

Language：English   Publishing type：Research paper (scientific journal)  

Chondroitin sulfate (CS) has been suggested to be involved in bone formation and mineralization processes. A previous study showed that squid-derived CS (sqCS) has osteoblastogenesis ability in cooperation with bone morphogenetic protein (BMP)-4 in vitro. However, in vivo, osteogenic potential has not been verified. In this study, we created a critical-sized bone defect in the rat calvaria and implanted sqCS-loaded gelatin hydrogel sponges (Gel) into the defect with or without BMP-4 (CS/BMP/Gel and CS/Gel, respectively). At 15 weeks, bone repair rate of CS/Gel-treated defects and CS/BMP/Gel-treated defects were 47.2% and 51.1%, respectively, whereas empty defects and defects with untreated sponges showed significantly less bone ingrowth. The intensity of von Kossa staining of the regenerated bone was less than that of the original one. Mineral apposition rates at 9 to 10 weeks were not significantly different between all treatment groups. Although bone repair was not completed, sqCS stimulated bone regeneration without BMP-4 and without external mesenchymal cells or preosteoblasts. Therefore, sqCS is a promising substance for promotion of osteogenesis.

DOI： 10.3390/md11125024

Language：English   Publishing type：Research paper (scientific journal)  

The tapetum lucidum is a light-reflective tissue in the eyes of many animals. Many ungulates have a fibrous tapetum. The horse has one of the largest eyes of any living animal and also has excellent vision in low-light environments. This study aimed to clarify the macroscopic tapetal shape, relationship between the tapetal thickness and the degree of pigmentation of the retinal pigment epithelium (RPE), spatial relationship between the visual streak and the tapetum, and wavelength of the light reflected from the tapetum in the horse. Macroscopically, weak light revealed the tapetum as a horizontal band located dorsal to and away from the optic disc. The tapetum expanded dorsally as the illumination increased. The tapetal tissue consisted of lamellae of collagen fibrils running parallel to the retinal surface; these spread over almost the entire ocular fundus and were thicker in the horizontal band dorsal to the disc. Only the horizontal band of the tapetum was covered by unpigmented RPE, suggesting that this band reflects light and is responsible for mesopic and scotopic vision. The visual streak was located in the ventral part of the horizontal band, ventral to the thickest part of the tapetum. The wavelength of the light reflected from the horizontal band of the tapetum was estimated from the diameter and interfibrous distance of the collagen fibrils to be approximately 468 nm. Therefore, the light reflected from the tapetum should be more effectively absorbed by rods than by cones, and should not interfere with photopic vision.

DOI： 10.1111/joa.12100

Language：Others  

Effectiveness of isoprothiolane in preventing fatty liver in dairy cows

Language：English   Publishing type：Research paper (scientific journal)  

The vasculature of the hemal node (HN) from the bovine cervical region was investigated using a combination of vascular corrosion casting and scanning electron microscopy. A dense vessel network of capsule was found surrounding the cast of HN parenchyma and had no connection with the subcapsular sinus; these vessels converged and exited the HN via the hilar vein. Within the HN, many anastomoses were found between the capillary networks and the surrounding sinuses in the follicular zone and deep cortex. The sinusoid pathway in the HN was characterized by subcapsular sinuses, which were continuous with the trabecular sinuses and tubular sinuses over the parenchyma, and these sinuses finally entered the medullary veins. In our study, direct communications between cortical capillaries and subcapsular sinuses were identified. This may explain the origin of numerous erythrocytes in the HN sinusoids and help to understand lymphocyte migration of the HN.

DOI： 10.1292/jvms.12-0156

Language：English   Publishing type：Research paper (scientific journal)  

The aim of this study was to clarify the effects of myostatin, which is a negative regulator of skeletal muscle mass, on the proliferation of NIH3T3 fibroblasts and the synthesis of extracellular matrix (ECM) by them. A proliferation assay revealed that myostatin attenuated cell growth at any of the doses used. High doses of myostatin strongly inhibited cell proliferation. Moreover, myostatin receptor, activin receptor type-2B (ActRIIB), was found to be distributed on cells and it was also clarified that myostatin increased the expression of cyclin-dependent kinase inhibitor p21 (p21). These results suggested that a high dose of myostatin inhibits fibroblast proliferation by the same mechanism as that for inhibition of myoblast proliferation. We then examined the effects of myostatin on the mRNA expression of ECM molecules (decorin, biglycan, type I collagen, type III collagen, type IV collagen and type V collagen) by real-time PCR. Real-time PCR showed that myostatin increased the mRNA of decorin, biglycan and collagen (types I, IV and V) in fibroblasts. The results suggest that myostatin regulates ECM synthesis in cultured fibroblasts.

DOI： 10.2220/biomedres.33.355

Language：English   Publishing type：Research paper (scientific journal)  

Vasopressin-induced intracellular Ca²⁺ concentration responses in non-neuronal cells of the rat dorsal root ganglion.
Arginine-vasopressin (AVP) is a nonapeptide of hypothalamic origin that has been shown to exert many important cognitive and physiological functions in neurons and terminals of both the central and peripheral nervous system (CNS and PNS). Here we report for the first time that AVP induced an increase in intracellular Ca²⁺ concentration ([Ca²⁺](i)) in non-neuronal cells isolated from the rat dorsal root ganglion (DRG) and cultured in vitro. The ratiometric [Ca²⁺](i) measurements showed that AVP evoked [Ca²⁺](i) responses in the non-neuronal cells and these concentration-dependent (100 pM to 1 μM) responses increased with days in vitro in culture, reaching a maximum amplitude after 4-5 day. Immunostaining by anti-S-100 antibody revealed that more than 70% of S-100 positive cells were AVP-responsive, indicating that glial cells responded to AVP and increased their [Ca²⁺](i). The responses were inhibited by depletion of the intracellular Ca²⁺ stores or in the presence of inhibitors of phospholipase C, indicating a metabotropic response involving inositol trisphosphate, and were mediated by the V₁ subclass of AVP receptors, as evidenced by the use of the specific blockers for V₁ and OT receptors, (d(CH₂)₅¹,Tyr(Me)²,Arg⁸)-Vasopressin and (d(CH₂)₅¹,Tyr(Me)²,Thr⁴,Orn⁸,des-Gly-NH₂⁹)-Vasotocin, respectively. V(1a) but not V(1b) receptor mRNA was expressed sustainably through the culture period in cultured DRG cells. These results suggest that AVP modulates the activity of DRG glial cells via activation of V(1a) receptor.

DOI： 10.1016/j.brainres.2012.08.028

Language：English   Publishing type：Research paper (scientific journal)  

The objective of this study was to investigate hepatocyte apoptosis in dairy cows during the transition period. Four clinically healthy, pregnant dairy cattle were used. The cows had no clinical diseases throughout this study. Blood samples were collected and livers were biopsied from the cows at 3 different times: 3 weeks before expected partition (wk -3); during parturition (wk 0), and 3 weeks (wk +3) after parturition. The damage to deoxyribonucleic acid (DNA) caused by hepatocytes was evaluated by comet assay. The apoptotic features of hepatocytes were examined by immunohistochemistry and electron microscopic analyses. The hepatic triglyceride content markedly increased at wk 0 and wk +3 compared with the values at wk -3. The results of the comet assay showed increases in the mean tail moment values of hepatic cells after parturition in all cows, which suggested increased DNA damage. Histopathologically, the hepatocytes began to contain lipid droplets at wk 0 and were severely opacified at wk +3. Caspase-3-positive and single-stranded DNA-(ssDNA)-positive cells were first detected in the liver after parturition. Condensation of nuclear chromatin, a typical sign of apoptosis, was confirmed by transmission electron microscopy after parturition. These results suggest that apoptosis is induced in hepatocytes of dairy cows around parturition and may result from lipotoxicity in hepatocytes.

Language：English   Publishing type：Research paper (scientific journal)  

Cerebellar folia may increase in number in hypothyroid rats (Lauder et al., 1974; Hasebe et al., 2008a). In this study, we aimed to confirm the formation of an excess sublobule and to determine whether excess sublobules are consistently formed in conserved positions in hypothyroid rats. Instead of the foliation pattern partitioned by cerebellar fissures, we employed the bifurcation pattern of the internal granular layer for investigation of complexity of the cerebellar cortex in hypothyroid rats. The basic foliation pattern of the cerebellum was intact in hypothyroid rats, but lobules III to IX frequently showed an increase in the number of sublobules. The excess sublobules were mainly found in the folia and along the shallow region of the fissure. In other words, the excess sublobules were not located in random locations but rather in specific locations. The area in the internal granular layer of lobules V to IX was significantly larger than that in control rats. From the increased area of the internal granular layer it may be inferred that internal granular cells increase in number than those in normal rats. In our study, regions within the cerebellum that show an excess of sublobules correlate with regions that show an intermediate to late-forming internal granular layer (Altman, 1969). Our observations fit with the view that excess sublobules are formed by the external granular layer showing prolonged cell proliferation and hypothyroidism predominantly has an adverse impact on the intermediate to late phases in development of the internal granular layer.

DOI： 10.1016/j.aanat.2011.12.005

Language：English   Publishing type：Research paper (scientific journal)  

The distribution and population of immunocompetent cells in bovine hemal node, mesenteric lymph node and spleen were analyzed comparatively by immunohistochemistry and flow cytometry. Many CD8(+) cells, CD172a(+) cells and γδ T cells were found in the lymphatic cord along the sinus of the hemal node and the splenic red pulp. A few CD8(+) cells and γδ T cells were distributed diffusely in the paracortex and medullary cord of the mesenteric lymph node. Many germinal centers were recognized in the lymphatic regions such as the cortex and white pulp of these lymphoid organs. The populations of CD8(+) cells and γδ T cells in the hemal node and the spleen were higher than those of the mesenteric lymph node. In addition, the populations of CD21(+) cells and MHC class II(+) cells in the hemal node and the mesenteric lymph node were higher than those of the spleen. The results suggest that the hemal node has an important role in both cellular and humoral immunity as well as the lymph node and the spleen in cattle.

DOI： 10.1292/jvms.11-0405

Language：English   Publishing type：Research paper (scientific journal)  

The aim in this study is to elucidate the laterality of chicken spinocerebellar (SC) neurons that originate from the caudal cervical to caudal lumbosacral spinal cord. SC neurons in the spinal segment (SS) 17-20 consisted of a mixture of crossed and uncrossed axons. SC neurons in the more cranial and caudal SS than SS 17-20 (transitional zone) were generally uncrossed and crossed, respectively. In the transitional zone, SC neurons in spinal border cells and ventral border cells of the ventral horn changed dramatically from an uncrossed to a crossed type between SS 17 and SS 18. Chicken SC neurons are markedly different in laterality from mammalian SC neurons.

DOI： 10.1292/jvms.11-0398

Language：English   Publishing type：Research paper (scientific journal)  

The ovary is the main secretory source of progestin and estrogen and is indispensable to the maintenance of all events of pregnancy in mice. The purpose of this study was to control all processes of pregnancy in mice, from embryo implantation to parturition, without ovaries. The ovaries were removed before embryo implantation, and a single injection of medroxyprogesterone acetate (MPA) was given. Embryo implantation was induced by leukemia inhibitory factor, which can substitute 17β-estradiol (E(2)). Continuous exposure to E(2) was necessary at mid-pregnancy, when placentation was completed. All mice sustained pregnancy without ovaries before parturition, which was initiated by the removal of E(2) and MPA. Murine pregnancy is a complicated process involving embryo implantation, placentation, and parturition. Complete control of pregnancy was achieved with the simple treatment of MPA and E(2) after induction of embryo implantation. Here, time-dependent events in the uterus during pregnancy could be realized without the ovaries, because the initiation of each event could be stringently controlled by hormonal treatments.

DOI： 10.1530/REP-11-0288

Language：English   Publishing type：Research paper (scientific journal)  

The equine superficial digital flexor tendon (SDFT) has a graded distribution of collagen fibril diameters, with predominantly small-diameter fibrils in the region of the myotendinous junction (MTJ), a gradual increase in large-diameter fibrils toward the osteotendinous junction (OTJ), and a mixture of small- and large-diameter fibrils in the middle metacarpal (MM) region. In this study, we investigated the ultrastructure of the SDFT, to correlate the spatial relationship of the collagen fibrils with the graded distribution. The surface-to-surface distances of pairs of fibrils were found to be almost constant over the entire tendon. However, the center-to-center distances varied according to fibril diameter. Decorin is the predominant proteoglycan in normal mature tendons, and has one dermatan sulfate (DS) or chondroitin sulfate (CS) filament as a side chain which is associated with the surfaces of the collagen fibrils via its core protein. We identified a coordinated arrangement of decorin DS filaments in the equine SDFT. The sizes of the decorin DS filaments detected by Cupromeronic blue staining showed a unique regional variation; they were shortest in the MM region and longer in the MTJ and OTJ regions, and a considerable number of filaments were arranged obliquely to adjacent collagen fibrils in the MTJ region. This regional variation of the filaments may be an adaptation to lubricate the interfibrillar space in response to local mechanical requirements. The results of this study suggest that the MTJ region, which receives the muscular contractile force first, acts as a buffer for mechanical forces in the equine SDFT. © 2011 The Authors. Journal of Anatomy © 2011 Anatomical Society.

DOI： 10.1111/j.1469-7580.2011.01456.x

Language：English   Publishing type：Research paper (scientific journal)  

KASEA16(+) and KASEA16(-) peptides, the net charges of which are positive and negative, respectively, under a neutral condition could undergo self-assembly into nanofibers and form transparent hydrogels without peptide aggregation upon rapid pH neutralization. The numbers of NIH3T3 cells attached to the KASEA16(+) hydrogel and KASEA16(-) hydrogel were similar, and cells proliferated with time on both hydrogels. Cells on the KASEA16(+) hydrogel had spindle-like morphology, while cells on the KASEA16(-) hydrogel formed clusters without extending cytoplasmic processes. Comparison of differently charged peptides under a neutral condition suggested that the charges of the scaffolds should be taken into consideration for the best design and selection of scaffolds for cell culture. Since the KASEA16(+) peptide could form a stable hydrogel under a neutral condition and the hydrogel served as a scaffold for cell proliferation, the KASEA16(+) hydrogel will be a useful scaffold for cell culture.

DOI： 10.1177/0885328210379927

Language：English   Publishing type：Research paper (scientific journal)  

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 µg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

DOI： 10.1262/jrd.11-048h

Language：English   Publishing type：Research paper (other academic)  

Language：English   Publishing type：Research paper (scientific journal)  

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (ip) injections with anti-LIF antibody (7.5 μg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after ip injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation. © 2012 Society for Reproduction and Development.

DOI： 10.1262/jrd.11-048H

Language：English   Publishing type：Research paper (scientific journal)  

The aim of this study was to determine the developmental changes of small leucine-rich proteoglycans (PGs), decorin, biglycan and fibromodulin, in ICR mouse retinas and to elucidate their role in the adult retina using kainic acid (KA)-induced retinal degeneration model. Retinas of prenatal, postnatal and adult mice were collected for histological and immunohistochemical staining to investigate the changes in distribution of these PGs. Decorin-and fibromodulin-immunostainings were diffusely distributed at prenatal and early postnatal stages and were stronger in the adult retina. However, biglycan was moderately distributed in the prenatal and early postnatal stages and was faint in the adult retina. Retinas were collected at 1, 3 and 7 days after intravitreal injection of KA. Retinas of KA injected eyes underwent shrinkage accompanied by serious damage in the inner layers. Decorin and fibromodulin were upregulated in the inner retinal layers of KA-injected eyes compared to the normal ones. Our results suggest that decorin and fibromodulin play key roles in retinal differentiation, and contribute to the retinal damage and repair process. However, biglycan may have no or only a limited role in the mouse retinal development or repair process.

DOI： 10.1292/jvms.10-0464

Language：English   Publishing type：Research paper (scientific journal)  

The constitution of ependyma derived from the ventricular zone is different from that derived from other regions of the central nervous system. In the mammalian cerebrum, the ependyma is varied by the regions to cortex or basal ganglia (BG). In the avian telencephalon (Tc), previous studies about the constitution of the ependyma have not revealed clear findings. In the present study, we performed immunostaining of ependymal cells in the chicken Tc to confirm differences in the ependyma of various regions. As a result, 4 patterns of ependyma were defined in the outer side of the lateral ventricle. In the base of the lamina pallio-subpallialis (LPS), ependyma consisted of vimentin/glial fibrillary acidic protein (GFAP) double-positive cells, whereas in the base of the lamina frontalis superior, it consisted primarily of vimentin-positive cells and a small number of vimentin/GFAP double-positive cells. With the exception of the above, the pallial ependyma was a single layer containing vimentin single-positive cells. Lastly, the ependyma of the BG was rich in vimentin single-positive cells. The constitutional differences of the ependyma of the pallium and BG concerned differences in ependymal morphology and cell characteristics. These finding suggest that the bounder between pallium and BG is LPS at the point of ependyma.

DOI： 10.1292/jvms.10-0377

Language：English   Publishing type：Research paper (scientific journal)  

The aim of the present study was to evaluate the relationship between the retention of fetal membranes (RFM) and apoptosis of the cells in fetal membranes. The present study investigated mRNA and protein expressions of apoptosis-regulating factors: FAS, cellular FLICE-like inhibiting protein (cFLIP), BAX, BCL2, caspase-8 (CASP8), and CASP3 in fetal membranes. Placentomes were manually collected from the uterus immediately after parturition and classified into two groups (RFM; n = 8 and non-RFM; n = 8) according to whether placental membranes were expelled or not within 12 h after delivery. FAS mRNA expression in maternal placental tissue was less in RFM cows than in non-RFM cows (P < 0.05). cFLIP mRNA expression in maternal and fetal placental tissue was greater in RFM cows than in non-RFM cows (P < 0.05). CASP3 mRNA expression in maternal placental tissue was greater in RFM cows than in non-RFM cows (P < 0.05). However, the protein expressions of FAS, cFLIP and cleaved CASP3 were not significantly different between the two groups. mRNA and protein expressions of BAX, BCL2 and CASP8 were also not significantly different between the two groups. In the immunohistochemical study, single-stranded DNA, which appears specifically in the apoptotic cells, was mainly found in the maternal placenta of non-RFM cows. Together these results suggest that RFM occurs at least in part due to a dysfunctional apoptotic process caused by the inhibition of FAS expression in the maternal placenta, and the increase of cFLIP expression in the maternal and fetal placenta.

DOI： 10.1016/j.placenta.2010.10.016

Language：English   Publishing type：Research paper (scientific journal)  

The aim of the present study was to determine the distribution of chondroitin sulfate proteoglycans in the mouse retina and optic nerve of the prenatal and postnatal mouse by immunohistochemistry. At embryonic day (E) 18, chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S) and biglycan were detected in the retina and optic nerve. However, aggrecan was seen in the retina but not in the optic nerve. At postnatal day (P) 7, aggrecan and biglycan were clearly observed in the optic nerve, inner nuclear layer and ganglion cell layer and diffuse in the outer retina. C4S diffusely distributed in the retina and optic nerve, but C6S was mainly confined to the photoreceptor layer and optic nerve sheath. At P42, biglycan showed diffuse distribution in the retina and optic nerve with intense staining in nerve-fiber rich layers. Aggrecan showed weak staining at the inner plexiform layer with higher density in the outer and inner nuclear layers, outer plexiform layer and ganglion cell layer. Both C4S and C6S were detected in the optic nerve and retina, but C6S showed strong immunostaining in the photoreceptor layer. The distributions of these proteoglycans with respect of time course during development of the retina and optic nerve suggest that they may have unique or overlapping roles in development and maintenance of the retina and optic nerve.

DOI： 10.1292/jvms.10-0201

Language：English   Publishing type：Research paper (scientific journal)  

The largest area of the avian telencephalon (Tc) is the subpallium [basal ganglia (BG)], and the pallium (cortex) is a narrow area located at the surface of the Tc. However, recent studies have proposed that most of the area of the avian Tc is the pallium, which corresponds to the cerebral cortex of mammals. This theory is based on neuronal elements with little regard to glial cells, which play important roles in neurogenesis. In the present study, we observed the distribution of glial cells using immunohistochemistry during maturation and discuss the division of the Tc by glial elements. In the early stage, the distribution and morphology of vimentin-positive radial glial cells were different between dorsal and ventral areas when they began to spread their processes toward the pia matter. During the development stage, vimentin-positive long processes divide the pallium and BG by the lamina pallio-subpallialis. Moreover, the pallium was divided into four regions by vimentin and glial fibrillary acidic protein-positive elements in the later stage.

DOI： 10.1292/jvms.10-0209

Language：English   Publishing type：Research paper (scientific journal)  

The arrangement of collagen fibrils and glycosaminoglycans (GAGs) in substantia propria are important for maintaining transparency of the cornea. Interferences in collagen fibrils and GAG production could be adversative to corneal integrity. In this study, six dogs consisting of four Beagles with normal cornea (normal), one Beagles with opaque cornea (sample No. 1) and one Shih Tzu with neovascularization opaque cornea (sample No.2) were used. All samples were observed morphologically by light and electron microscopes to obtain diameter and distribution of collagen fibrils in substantia propria and were performed biochemically to investigate into GAGs and collagen types. The average diameter of collagen fibrils in the intact cornea of normal, sample No.1 and No.2 was 33.2, 35.0 and 25.0 nm, respectively. The percentage of matrix per unit area was 67% in normal, 87% in sample No.1 and 28.3% in sample No.2. The type III collagen ratio was 25.3% in normal, 21.3% in sample No.1 and 35.8% in sample No.2. The relative amount of heparan sulfate, chondroitin sulfate, dermatan sulfate and keratin sulfate was 1.5, 9.7, 51.1 and 37.7% in normal, 3.3, 26.0, 45.7 and 23.7% in sample No.1 and 1.2, 18.0, 16.6 and 54.1% in sample No.2. Hyaluronic acid was found only in sample No.1 with a relative amount of 1.3%. Since there was some relationship between collagen formation and GAGs composition, it might be speculated that disturbance in arrangement of collagen fibrils and GAG metabolism especially in substantia propria would bring up opacity of the cornea.

DOI： 10.2535/ofaj.87.59

Language：English   Publishing type：Research paper (scientific journal)  

Retinal topographies of some cell types and distribution of the tapetum lucidum in the sheep's eye were investigated in this study. The tapetum was observed macroscopically in the fundus. The topographical distributions of retinal ganglion cells (RGCs), cones, and rods were simultaneously analyzed in retinal whole mounts stained with cresyl violet. Short-wavelength-sensitive (S) cones were immunocytochemically identified in retinal whole mounts. The tapetum was located dorsal to the optic disc, with the nasal part elongated horizontally and the temporal part expanded dorsally. RGCs were distributed densely in the area centralis, horizontal visual streak, and anakatabatic area. The highest density in the area centralis was approximately 18,000 RGCs/mm(2). Cones showed high density in the horizontal area crossing the optic disc and dorsotemporal area, whereas rods showed high density in the horizontal area, which was greater in height than the horizontal area of high cone density. S cones showed high density in the dorsotemporal retina. The rod/cone ratios were high horizontally in the dorsal retina to the optic disc, with a mean value of 11:1. The cone/RGC and rod/RGC ratios were lower in the horizontal and dorsotemporal retina, and the rod/cone/RGC ratio was lowest in the area centralis (9:1:1). The retinal topographies and distribution of the tapetum were specialized in the horizontal and dorsotemporal fundus. This suggests that sheep have better visual acuity in horizontal and anteroinferior visual fields and that this specialization is related to the visual ecology of sheep. J. Comp. Neurol. 518:2305-2315, 2010. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/cne.22333

Language：English   Publishing type：Research paper (scientific journal)  

The aim of this study was to determine the characteristic differences in tendinocytes derived from tendons in the equine forelimb, superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET), in morphology, proliferation, collagen production ability and ability for synthesis of matrix metalloproteinases (MMPs). Significant differences were observed in cell number in vivo. The cellular number was largest in the SDFT and smallest in the CDET. The values of in vitro proliferation ratios and ability for synthesis of collagen and MMPs were largest in the SDFT and smallest in the CDET. Addition of TNFalpha to culture of all three types of tendinocytes increased the synthesis of both proMMP-9 (except CDET) and collagen and decreased proMMP-13 synthesis and had no effect on proMMP-2 synthesis. Flexor tendons in forelimbs (SDFT and DDFT) restore energy during locomotion and are more easily injured than are extensor tendons. This structural property would cause active ECM and MMPs synthesis. And CDET have very low turnover potential; in the small number of cells, low cellular proliferation, lower ability for synthesis of collagen and MMPs. The isolated tendinocytes provided much information on the characteristics and properties of tendons for the ECM turnover system and responsiveness of tendinocytes to complex inflammatory responses in tendinopathy.

DOI： 10.1016/j.tice.2009.06.002

Language：English   Publishing type：Research paper (scientific journal)  

The tapetum lucidum is a light-reflective device improving visual sensitivity in mesopic vision. There have been few studies on tapetal distribution and its relationship with degree of pigmentation in the retinal pigment epithelium (RPE). In the present study, sheep's eyes were used for macroscopical observation of the tapetum, and then histological sections of the posterior eyecups were made to analyze the distribution of tapetal thickness and degree of pigmentation in the RPE. Macroscopically, with available light, the tapetum was located in the dorsal eye fundus to the optic disc and showed an L-shape with the horizontally elongated nasal part and the dorsally expanded temporal part. In photographs with a flash, the tapetal area expanded and showed a more triangular shape. The tapetum histologically consisted of layers of dense collagen fibers and was thicker in the temporal part than in the nasal part. The maximum tapetal thickness was approximately 70 pm. The histological tapetal area was similar to the tapetal shape with a flash light. The pigmentation of the RPE was divided into three types, nonpigmented, transitional, and pigmented areas. The nonpigmented area was similar to the tapetal shape with available light. It is suggested that approximately 55% of the histological tapetal area is covered with the nonpigmented area and is functional under a natural light condition. The functional tapetal area was similar to the L-shaped high-density area of retinal ganglion cells.

DOI： 10.1292/jvms.09-0413

Language：English   Publishing type：Research paper (scientific journal)  

This aim of this study was to determine the characteristic differences in tendinocytes derived from three sites of the equine superficial digital flexor tendon (SDFT)-proximally the myotendinous junction (MTJ), mid-metacarpal (mM) and osteotendinous junction (OTJ)-in morphology, proliferation, and ability for synthesis of collagen and matrix metalloproteinases (MMPs). Little difference was observed in cell proliferation. Addition of tumor necrosis factor (TNF) alpha to the culture medium resulted in increased collagen synthesis by tendinocytes from all three sites. The amount of collagen synthesized by tendinocytes derived from the mM and OTJ was much larger than that synthesized by untreated tendinocytes. A collagen zymogram revealed that proMMP-13 synthesis was increased towards the distal site. However, TNFalpha treatment resulted in a significant decrease in the amount of proMMP-13 synthesized by tendinocytes from all three sites. On the other hand, a gelatin zymogram showed that the synthesis level of proMMP-9 tended to decrease towards the distal site, but there was little difference between synthesis levels of proMMP-9 before and after TNFalpha treatment. These results indicated that tendinocytes in the same tendon have different characteristics and that these characterisities would reflect the function of tendinocytes in vivo. Also, the isolated tendinocytes provided much information on the characteristics and properties of tendons for the ECM turnover system and on the responsiveness of tendinocytes to complex inflammatory responses in a tendinopathy condition.

DOI： 10.2220/biomedres.31.35

Language：Japanese  

Morphological changes of the jejunum villi to di erent diets
The aim of this study was to evaluate morphological responses of the absorptive apparatus of the jejunum to different diet ingredients in weaning pigs. In this study, seven crossbred (Landrace&times;Large Yorkshire&times;Duroc) pigs, they were a litter, were used after weaning. Before weaning, they fed with creep feed for six days. Six pigs were divided into two groups ; one received dairy-based feed and the other received vegetable-based feed. The remaining pig was used as a non-treatment control. Dairy-based feed contained mainly animal-based feed, and vegetable-based feed contained mainly oil-seed cake and meal. Drinking water was provided ad libitum throughout the experimental period. Jejunum samples were collected immediately after weaning from a non-treated control pig (D0). The similar samples were collected from each 3 pigs of dairy-based feed and vegetable-based feed groups respectively, on 3 (D3) and 14 days (D14) after weaning. The samples were processed for morphological observation of absorptive epithelial cells and heights of villi and microvilli. A significant increase in height of intestinal villi on D3 and in size of the absorptive cells on D14 were found in animals that received vegetable-based feed , ultrastructural transition of epithelial cells on D3. An increases in microvilli height on D3 and D14 were seen in animals that received vegetable-based feed. Presumably, the ingredients of vegetable-based feed are more easily digested and absorbed through the intestinal wall than are ingredients of dairy-based feed. As a result, the growth of intestinal villi and (growth of) absorptive epithelial cells in vegetable-based feed group were greater than those in dairy-based feed group, leading to a more efficient absorptive function of the small intestine.

DOI： 10.2508/chikusan.80.171

Language：English  

Efficacy of Antivascular Photodynamic Therapy Using Benzoporphyrin Derivative Monoacid Ring A (BPD-MA) in 14 Dogs with Oral and Nasal Tumors
Antivascular photodynamic therapy (PDT) Suppresses tumor growth and prolonged the survival in solid tumor-bearing mice. The purpose of this study was to assess the efficacy of antivascular PDT using BPD-MA for treatment of oral and nasal tumors in 14 dogs. At 15 min after initiating intravenous infusion of 0.5 mg/kg benzoporphyrin derivative monoacid ring A, tumors were irradiated with laser light at 690 nm emitted by a diode laser, The 1-year survival rate of 7 dogs with oral tumors was 71%. The 1-year Survival rate of 7 does with nasal tumors was 57%. Imaging of each tumor was performed by using angiographic computed tomography before and after each antivascular PDT. Contrast-enhanced turners were observed before antivascular PDT, but these tumors were not enhanced with contrast medium following antivascular PDT. Antivascular PDT is suggested to be a promising method for dogs with oral and nasal tumors that cannot be effectively treated with current antitumor therapies.

DOI： 10.1292/jvms.71.125

Language：English   Publishing type：Research paper (scientific journal)  

Effect of Collagen Oligopeptide Injection on Rabbit Tenositis
Effects of the collagen oligopeptide (COP) were examined by its repeat injection into the inflammatory rabbit Achilles tendon (Tendo calcaneus communis), in which tenositis was induced by injection of bacterial collagenase. COP was evaluated 5 times over a period of 3 weeks to I month after injection of collagenase. At I month after treatment, the therapeutic effect of COP was evaluated by examining the structure of collagen fibrils, amount and components of glycosaminoglycans (GAGS) and matrix metalloproteinases (MMPs), and compared with the saline injection, control, and normal groups. The Achilles tendon of rabbit in the control group (no COP injection) and saline injection group showed a notable increase in the number of fine collagen fibrils, a change in GAG composition and increase in the amount of pro-MMP-2, indicating the weakening of the tendon. In contrast, the size distribution of collagen fibrils, GAG composition and the amount of pro-NIMP-2 was similar to those in the normal group. These results suggest that COP injection promotes healing processes of the tendon injury.

DOI： 10.1292/jvms.70.1295

Language：Japanese  

Morphology of mesenteric arteries in horse having endotoxemia-related colic
ウマにおいて疝痛を伴った内毒素血症はしばしば発症する疾病であって、毒素によって腸間膜動脈には様々な程度の虚血、動脈の痙攣や壊死が引き起こされる。重篤な場合では、それらの臨床症状が疝痛の前駆的発症要因になることが報告されている。エンドトキシンが関与し疝痛を患ったウマでは、腸間膜の血管における障害はこれまでのところ大いに注目されてきている。そこで、この研究では内毒素血症を自然発症したサラブレットにおいて、腸間膜動脈の障害を形態学的に検索した。この研究で得られた結果は、腸間膜にみられる血管の組織構造に対するエンドトキシンの効果をより良く理解できるものと思われる。試料は3.0％グルタルアルデヒド溶液に固定後、Quetol 812に包埋した。包埋した電顕試料より厚切り切片および超薄切片を作製し、前者には1％トルイジンブルー染色を施した後、光学顕微鏡を用いて、後者には酢酸ウラニウムとクエン酸鉛による二重染色を行った後、透過型電子顕微鏡にて詳細に検索した。発症群では動脈壁において血管内皮細胞の剥離、出血像、歪な形を呈した中膜平滑筋細胞や融解壊死した顆粒球などの血液細胞、空胞などが多数観察された。これらの結果は、すでに報告されているウマの内毒素血症の組織像と類似しており、また、エンドトキシンにより腸間膜中の動脈が損傷を受け、ウマの腸管において臓器性Shwartzman反応が起こったものと示唆された。

Language：English   Publishing type：Research paper (scientific journal)  

Morphological Analysis of Corneal Opacity in Shiba Dog with GM1 Gangliosidosis
GM1 gangliosidosis is one of the inherited metabolic lysosomal storage disorders characterized by neurological symptoms caused by beta-galactosidase deficiency and consequent accumulation of GM1 ganglioside in neuronal cells. Shiba dogs affected with GM1 gangliosidosis have been found to suffer from corneal opacity. In our morphological analysis, keratocyte enlargement was induced by abnormal intracellular accumulation of neutral carbohydrates, resulting in the loss of normal arrangement of collagen fibrils in the opaque cornea was found to be associated with the disorder. We therefore conclude that corneal opacity in this Shiba dog with GM1 gangliosidosis may be caused by neutral carbohydrate accumulation in lysosomes, swelling and dysfunction of keratocytes, and subsequent irregular arrangement of collagen fibrils in the corneal proper substance.

DOI： 10.1292/jvms.70.881

Language：English   Publishing type：Research paper (scientific journal)  

The modulation of collagen fibril assembly and its structure by decorin: An electron microscopic study
The present study was carried out to determine the effect of decorin in the process of collagen assembly. Collagen fibrils were obtained in vitro by aggregation from commercialized acid-soluble type I collagen with the addition of different concentrations of decorin (0-25 mu g/ ml). All specimens were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The distribution of collagen fibril diameters was also analyzed by TEM. In samples without or with low concentrations of decorin, highly porous collagen fiber networks were formed. On the other hand, dense networks were observed in samples treated with high concentrations of decorin. The influence of decorin secreted by cells on collagen fibrils was observed by SEM, and the fiber network elasticity was measured using a rheometer. SEM images showed that collagen fiber networks without fibroblasts were much looser than those cultured with normal fibroblasts. The networks cultured with the fibroblasts were composed of straight fibers with large diameters. On the other hand, collagen fiber networks cultured with siRNA-decorin-transfected (siDT) fibroblasts had loose, meandering fibers with small diameters. The elasticity of collagen fiber networks cultured with untransfected fibroblasts showed no significant difference over the 7-day incubation period. However, significantly lower elastic values were obtained for coliagen fiber networks treated with siDT cells on days 3 and 7. In addition, after treatment with 5.0 or 25 mu g/ml decorin, the collagen fiber networks cultured with siDT cells exhibited an altered structure that showed a dense structure similar to that of the fiber networks cultured with untransfected fibroblasts. In conclusion, this in vitro study showed that decorin is a regulatory and architecturally small leucinerich repeat proteoglycan in the process of collagen fibril assembly.

DOI： 10.1679/aohc.71.37

Language：English   Publishing type：Research paper (scientific journal)  

The superficial digital flexor tendon (SDFT) is one of the longest tendons in the horse. In racehorses, disturbance of the locomotor functions of the SDFT occurs most frequently in the central area of the mid-metacarpal region. While many studies have investigated the equine SDFT, there are no reports to date of the morphological characteristics of collagen fibrils in the central and peripheral areas of each of the three regions that comprise the entire tendon: the myotendinous junction (MTJ), the mid-metacarpal region (mM) and the osteotendinous junction (OTJ). Mass average diameter (MAD), which provides functional information on the mean collagen fibril diameter and tensile strength of the tendon, was found to be smaller in the central area than in the peripheral area of all three regions. The MAD value was lowest in both the central and peripheral areas in the MTJ region, and tended to increase generally in a distal direction in the OTJ. The OTJ is important parts that unite with the bone. We conclude that morphological structure suggested that it corresponds to biomechanical function in some region of the equine SDFT.

DOI： 10.2535/ofaj.84.111

Language：English   Publishing type：Research paper (scientific journal)  

Active expression of matrix metalloproteinase-13 mRNA in the granulation tissue of equine superficial digital flexor tendinitis.
The DNA microarray analysis for matrix metalloproteinase (MMP)-related mRNA expression in equine superficial digital flexor tendinitis indicated that mRNA level of MMP-13 was apparently up-regulated in the tendinitis as compared to normal tendon. In situ hybridization also revealed that fibroblastic cells proliferated in the granulation tissue generated in the tendinitis actively expressed MMP-13 mRNA. On the other hand, in normal tendon, a few fibroblastic cells and vascular components lied in the endotenon barely expressed its mRNA, but other cellular components in the tendon bundle were not positively hybridized. As mentioned above, MMP-13 but not other collagenases or gelatinases, may play an important role in tendon injuries in the racehorses.

DOI： 10.1292/jvms.69.637

Language：English  

Antitumor effects and blood flow dynamics after photodynamic therapy using benzoporphyrin derivative monoacid ring A in KLN205 and LM8 mouse tumor models
Photodynamic therapy (PDT) using benzoporphyrin derivative monoacid ring A (BPD-MA) induces direct tumor cell damage and microvascular injury. We administered BPD-MA at 3 h or 15 min before laser irradiation to KLN205 and LM8 tumors in murine models. Tumor growth delay was induced more effectively by 15-min-interval PDT than by 3-h-interval PDT. Vascularity and blood perfusion was significantly decreased by 15-min-interval PDT. We observed death of all tumor cells, except peripheral cells, in the 3-h-interval PDT group, and death of cells around the damaged tumor vasculature in the 15-min-interval PDT group. Thus, 15-min-interval PDT enhanced the antitumor effect by damaging tumor vasculature.

Language：English   Publishing type：Research paper (scientific journal)  

A paracrystalline structure was observed within left ventricular cardiomyocyte nuclei of MLP-/- mice. The paracrystal possessed cross lines, similar to 8.0 mu m long and 0.3 mu m wide, with a slender spindle shape and a periodicity of 13 nm. Paracrystals were best observed along the longitudinal orientation of myofibrils and were detected in less than 10% of the nuclei observed. One dimension of the protein unit forming the paracrystal was 8.5 nm long. The electron density of the paracrystal appeared to be slightly higher than that of heterochromatin, suggesting that RNA-associated proteins are constituents of the paracrystal. This is the first report of intranuclear paracrystals in cardiomyocytes, which appear to be unique to MLP-/- mice.

DOI： 10.1002/jemt.20386

Language：English   Publishing type：Research paper (scientific journal)  

The aim of this study was to clarify whether matrix metalloproteinases (MMP-2 and -9: gelatinases) and pro-inflammatory cytokines [tumor necrosis factor (TNF) alpha and interleukin (IL)-1 beta] are induced by heat in tendon tissue in vitro and to test the hypothesis that heat exposure causes tendinocytes to synthesize pro-inflammatory cytokines and that synthesis of these cytokines, in turn, leads to up-regulation of synthesis of gelatinases. Isolated tendinocytes from equine superficial digital flexor tendons were cultured and all experiments were performed on cells passaged 3 or 4 times. In the cells exposed to heat (37 to 45 degrees C, 0 to 60 min), the survival rate decreased sharply in a temperature- and time-dependent manner, especially at 42 and 45 degrees C. Cells exposed at 40 degrees C, however, showed little change in survival rate and morphology. Gelatin zymograms revealed that proMMP-2 and -9 were the only two MMPs remaining in the supernatant of the cultured tendinocytes, including that of untreated cells. Addition of TNF alpha and IL-1 beta to the culture medium of tendinocytes accelerated proMMP-9 synthesis considerably. Heating the tendinocytes (40 degrees C) led to a three-fold increase in proMMP-9 synthesis in a short time. Only TNFa was detected in tendinocytes after heat exposure for 30 and 60 min. In contrast, IL-1 beta was under the detectable level in ELISA. Cooling of heat-exposed cells from 40 degrees C to 37 degrees C considerably down-regulated cellular proMMP-9 synthesis. Furthermore, proMMP-9 level was greatly reduced in cells treated at lower temperatures, 20 degrees C and 5 degrees C. These findings support our hypothesis that hyperthermia in the horse tendon induces tendinocytes to synthesize pro-inflammatory cytokines and that the synthesis of these cytokines results in the up-regulation of gelatinases.

DOI： 10.2220/biomedres.27.233

Language：English   Publishing type：Research paper (scientific journal)  

Effects of ingestion of collagen peptide on collagen fibrils and Glycosaminoglycans in the dermis
In order to investigate the effects of collagen peptide ingestion on fibroblasts and the extracellular matrix in the dermis, collagen peptide was administered orally to pigs at 0.2 g/kg body weight/d for 62 d, and its effects were compared with those of lactalbumin and water controls. Fibroblast density, and diameter and density of collagen fibrils were significantly larger in the collagen peptide group than in the lactalbumin and water control groups. The two major components of dermal glycosaminoglycans, hyaluronic acid and dermatan sulfate, which are present in the inter-fibrillar space, did not differ significantly among the three groups. However, the ratio of dermatan sulfate, which is derived from fibril-bound decorin, was largest in the collagen peptide group. These results suggest that ingestion of collagen peptide induces increased fibroblast density and enhances formation of collagen fibrils in the dermis in a protein-specific manner.

DOI： 10.3177/jnsv.52.211

Language：English   Publishing type：Research paper (scientific journal)  

The cruciate ligament and the collateral ligament play key roles in stabilization of the knee joint. Cases of serious knee joint problems presented at the, the Veterinary Teaching Hospital of Rakuno Gakuen University, Japan mostly involved rupture of the cranial cruciate ligament (CCL). Disorders in structural and biochemical components of the CCL were thought to be the causes of the knee problems. Morphological, biochemical and biomechanical features of the CCL and the lateral collateral ligament (LCL) were therefore analyzed. In the CCL, fibroblasts with ovoid and enlarged nuclei were observed mainly at the periphery of collagen bundles. The array of collagen fibrils in the LCL was slightly disoriented, but that of the CCL was tight and regular. In the LCL, the major groups of collagen fibrils were those with diameters of 70-80 and 120-130 nm. Most collagen fibrils in the CCL had diameters of 70-80 nm. The mean collagen diameters were 90 nm in the CCL and 105 nm in the LCL. The ratios of the noncollagen area to the area occupied by collagen fibrils were 43% in the CCL and 55% in the LCL. There was no difference between the amounts of HA or between the amounts of DS in two ligaments. However, the amount of CS in the CCL was about 17-times greater than that in the LCL. The expansion of and the resistance to tension exerted onto the CCL were less than those of the LCL. A high concentration of CS and low tensile strength due to small-sized collagen fibrils cause the CCL to rupture easily, especially when overextension of the knee joint occurs.

DOI： 10.2535/ofaj.83.25

Language：English  

The aim of this study was to investigate alterations of cultured tendon tissues to determine whether tissue culture is a useful method for biological analyses of the tendon. Tendon tissues for tissue culture were isolated from Achilles tendons of rabbits. The tendon segments were placed one segment per well and incubated in growth medium consisting of Dullbecco's modified Eagle's medium supplemented with 5% fetal bovine serum at 37 degrees C in a humidified atmosphere with 5% CO2 for various periods. The alignment of collagen fibrils was preserved for 48 h, but tendon structure has disintegrated at 96 h. Alcian blue staining and gelatine zymography revealed that proteoglycan markedly diminished and that matrix metalloproteinase (MMPs) activity was upregulated sharply at 72 and 96 h. The ratio of collagen fibrils with large diameter had increased and the mean diameter and mass average diameter value had reached maximum at 48 h. The values then decreased and mean diameters at 72 and 96 h were significantly different from that at 48 h. At 96 h, the ratio of collagen fibrils with small diameters had increased and collagen fibrils with large diameters had disappeared. These findings indicate that structural alteration is possible to be induced by disintegration of collagen fibrils and disappearance of glycosaminoglycans from extracellular matrix (ECM), subsequent of upregulation of MMPs activity. Although the study period is limited, the tissue culture method is available for investigating cell-ECM interaction in tendons.

DOI： 10.2220/biomedres.26.279

Language：English   Publishing type：Research paper (scientific journal)  

Differences in tumor necrosis factor (TNF)alpha and TNF receptor-1-mediated intracellular signaling factors in normal, inflamed and scar-formed horse tendons.
Tumor necrosis factor (TNF) receptors (TNF-R)-mediated cell survival or apoptosis has been demonstrated in many cells, but little is known about survival or apoptotic signals via TNF-R1 in tendinocytes. In this study, we focused on four signaling factors, TNFalpha, TNF-R1, TNFR-associated factor2 (TRAF2) and caspase-3, in order to elucidate the signaling events in tendinocytes. Samples were obtained from normal, inflamed and scar-formed equine superficial digital flexor tendons. To detect these signaling factors, samples were subjected to immunohistochemistry and Western blot analysis, and some samples were also subjected to reverse transcription-polymerase chain reaction (RT-PCR), PCR-Southern blot analysis and in situ hybridization to detect the expression of TNFalpha mRNA. Distribution of the four factors differed depending on the tendon condition, normal, inflamed or scar-formed. In the normal tendon, large amounts of TRAF2 were found in tendinocytes, but the amounts of TNF-R1 were small. TNFalpha mRNA was expressed most highly in the inflamed tendon. TNF-R1, which was only faintly detected in the normal tendon, was detected at a high level in the inflamed tendon, and the amounts of TRAF2 and caspase-3 also increased. Activated caspase-3 was only detected in the inflamed tendon. TNFalpha mRNA was also expressed in the scar-formed tendon, though it showed weak signals, and the expression levels of TNF-R1, TRAF2 and caspase-3 proteins were very low. Two distinct intracellular signaling pathways of TNFalpha, which lead to cell survival and apoptosis, might be present in tendinocytes mediated through TNF-R1. These results, which reflect the dynamism of TNFalpha, provide important clues for means to prevent tendinopathy.

DOI： 10.1292/jvms.67.985

Language：English  

Differences in tumor necrosis factor (TNF)alpha and TNF receptor-1-mediated intracellular signaling factors in normal, inflamed and scar-formed horse tendons
Tumor necrosis factor (TNF) receptors (TNF-R)-mediated cell survival or apoptosis has been demonstrated in many cells, but little is known about survival or apoptotic signals via TNF-R I in tendinocytes. In this study, we focused on four signaling factors, TNFU, TNF-R1, TNFR-associated factor2 (TRAF2) and caspase-3, in order to elucidate the signaling events in tendinocytes. Samples were obtained from normal, inflamed and scar-formed equine superficial digital flexor tendons. To detect these signaling factors, samples were subjected to immunohistochemistry and Western blot analysis, and some samples were also subjected to reverse transcription-polymerase chain reaction (RT-PCR), PCR-Southern blot analysis and in situ hybridization to detect the expression of TNF alpha mRNA. Distribution of the four factors differed depending on the tendon condition, normal, inflamed or scar-formed. In the normal tendon, large amounts of TRAF2 were found in tendinocytes, but the amounts of TNF-R I were small. TNFa mRNA was expressed most highly in the inflamed tendon. TNF-R1, which was only faintly detected in the normal tendon, was detected at a high level in the inflamed tendon, and the amounts of TRAF2 and caspase-3 also increased. Activated caspase-3 was only detected in the inflamed tendon. TNFa mRNA was also expressed in the scar-formed tendon, though it showed weak signals, and the expression levels of TNF-R I, TRAF2 and caspase-3 proteins were very low. Two distinct intracellular signaling pathways of TNF alpha, which lead to cell survival and apoptosis, might be present in tendinocytes mediated through TNF-R1. These results, which reflect the dynamism of TNF alpha, provide important clues for means to prevent tendinopathy.

DOI： 10.1292/jvms.67.985

Language：English   Publishing type：Research paper (scientific journal)  

The fate of iron (Fe) after bleeding has been analyzed in various connective tissues, but there have been only a few inconclusive studies on Fe in the tendon. In this study, energy dispersive X-ray microanalysis and an iron staining method were used to determine the localization of Fe in cells of the equine superficial digital flexor tendon. In inflamed tendons, Fe was detected in tendinocytes as well as macrophages. In contrast, negative or weak reactions were observed in many cells in the normal tendon. Although the accepted theory states that the reticuloendothelial system (macrophages and reticuloendothelial cells) is mainly responsible for the uptake and decomposition of erythrocytes, and tendon cells under conditions of inflammation show a functional ability to take up Fe substances. It has been reported that tendinocytes have multiple functions, including synthesis and secretion of collagen, cytokines, and matrix metalloproteinases. Taking these functions into consideration, indicates that the tendinocyte is not only an active cell but also a multi-functional cell.

DOI： 10.1679/aohc.68.161

Language：English  

Effects of Ingestion of Collagen Peptide on Collagen Fibrils and Glycosaminoglycans in Achilles Tendon
In order to investigate whether the oral ingestion of collagen peptide affects the extracellular matrix of tendon, two doses (0.2g/kg and 1.0g/kg body weight) were orally administered daily for 56d to a rabbit, and both the size of collagen fibrils and the amount of glycosaminoglycans in the Achilles tendon were measured in comparison with those in a rabbit fed with a control protein, lactalbumin, or water alone. Ingestion of col-lagen peptide or lactalbumin induced a significant increase in collagen fibril diameter and a decrease in fibril density except for a high dose of lactalbumin compared with the water con-trol. A histogram pattern of fibril diameter in a high dose of collagen peptide showed a peak at 160-180nm, which was not observed in other groups. However the percentage of diam-eters over 200nm was the lowest in this group but highest in the low-dose group of collagen peptide. The mean fibril diameter and mass average diameter of a high dose of collagen pep-tide were significantly smaller than those in a low dose. The amount of dermatan sulphate increased in the high-dose groups, while the amount of hyaluronic acid decreased in rabbits fed with collagen peptide or lactalbumin at either dose. These results suggest that the inges-tion of collagen peptide affects the size of collagen fibrils and composition of glycosaminogly-cans in the Achilles tendon and thus may improve the mechanical properties of the Achilles tendon.

DOI： 10.3177/jnsv.51.169

Language：English  

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5% and 91.9% homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation, migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.

DOI： 10.1292/jvms.67.385

Language：English  

Downregulation of Decorin and Transforming Growth Factor-β1 by Decorin Gene Suppression in Tendinocytes
Scars formed after tendonitis result in altered tissue mechanical properties after injury. The interaction of collagen molecules with decorin affects collagen fibrogenesis, and scar tissue is fragile as a consequence of a large amount of decorin in the scar. We hypothesized that scar formation could be prevented by controlling decorin expression in tendinocytes. As a preliminary experiment, we treated tendinocytes with decorin antisense oligodeoxynucleotides (ODNs). Tendinocytes were isolated from Achilles tendons of New Zealand white rabbits and treated with ODN. When tendinocytes were transfected with decorin sense ODN, there was no alteration, whereas decorin antisense ODN-treated tendinocytes showed suppression of transforming growth factor (TGF)-beta1 production. Decorin and TGF-beta1-production of tendinocytes is regulated by decorin gene suppression. The results showed that the antisense approach is an attractive therapeutic strategy not only for preventing decorin deposition in scar tissue, which decreases collagen fibril diameter, but also for controlling TGF-beta1 production, which leads to organ fibrosis.

DOI： 10.1080/03008200590935510

Language：English  

The aim of the present study was to clarify the presence and determine the role of apoptosis in the degenerative process of the superficial digital flexor tendon (SDFT) in the horse. Samples were obtained from normal and inflamed SDFTs of horses. To detect apoptosis and to identify apoptotic cells, the samples were subjected to immunohistochemical labelling and Western blot analysis. Although a large number of cells in degenerate areas showed positive reactions with caspase-3 and single stranded DNA antibodies, cells in normal tendon samples showed very weak reactions. Excessive apoptosis was confirmed by the results of Western blot analysis, which showed a significant increase in activated caspase-3 protein in the inflamed SDFTs, suggesting that apoptosis occurred in the tendinocytes via a caspase-3-dependent pathways This is the first report of excessive apoptosis in inflamed SDFT of the horse. The results indicate that apoptosis may play an important role in the process of degeneration of the tendon as well as other tissues. (C) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jcpa.2004.06.006

Language：English   Publishing type：Research paper (scientific journal)  

Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-R-associated factor 2 (TRAF2) and nuclear factor-kappa B (NF-kappaB), and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTs of thoroughbred horses. The tendinocytes were treated with 10 ng/ml equine TNFalpha medium for 6 hours and then the four factors on tendinocytes were visualized by using an immunohistochemical method, and the amounts of the four factors were determined by Western blot analysis. Although TNF-R1 and TNF-R2 co-localized on the same tendinocyte, in untreated control cells (normal condition), immunoreactivity against TNF -R1 was very weak but TNF-R2 showed a strong reaction. However, TNF-R1 showed the same high level of reaction as TNF-R2 in TNFalpha-treated cells (inflamed condition). Intense TRAF2 and NF-kappaB were detected at inflamed condition, however both factors were also detected at normal condition. The distinct distributions of the four factors under different conditions (normal and inflamed condition) in vitro not only reflect the dynamism of the cytokines but may also provide important clues for a means to prevent from occurrence of tendonitis and progress of tendon degeneration.

DOI： 10.14943/jjvr.52.3.135

Language：English  

The Golgi tendon organ (GTO) is an encapsulated fusiform mechanoreceptor siding in the musculo-tendinous junction of many animal species. Inhibitory function of afferent nerve fibers distributed from nearby motor units, the organ responds to active tension exerted onto the muscle. The morphological features of the equine GTO have not yet been elucidated. Additionally, there is some controversy regarding to the existence of the GTO in the equine superficial digital flexor tendon (SDFT). Therefore, immunohistochemistry and immunoelectron microscopy using alcian blue (pH 2.5) staining and the silver-enhanced colloidal gold method were carried out to determine both the location and characteristics of the GTO at the musculo-tendinous junction of the SDFT. A GTO with a fusiform structure of approximately 3 mm in length was found in the tendinous part. The lumen of the GTO was divided into compartments by septal cells. Each compartment contained collagen fibrils, nerve fibers and Schwann cells. This is the first report of the equine GTO.

DOI： 10.2535/ofaj.81.33

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The ultrastructure of Sertoli cell in the testes of Korean native goats was investigated by scanning and transmission electron microscopy to elucidate the relationship between germ cell and Sertoli cell processes in the spermiogenetic cycle. Type-A Sertoli cells at stage V of the spermiogenetic cycle and type-B Sertoli cells at stage VII of the spermiogenetic cycle were used for analysis. Morphologically, the Sertoli cell processes were classified into sheet-like and slender cord-like processes. The sheet-like process originated solely from the Sertoli cell column while the slender cord-like process projected either from the Sertoli cell column or the sheet-like process. Periodic acid (PA)-thiocarbohydrazide (TCH)-silver protein (SP)-physical development (PD)-positive granules were found diffusively both in the sheet-like and slender cord-like processes near the round spermatid, whereas they had accumulated near the head of the elongated spermatid. The morphological variation and glucoconjugate histochemical reaction of the Sertoli cell processes reflect nourishment, movement and transformation of the spermatogenic cells in accordance with spermiogenesis.

DOI： 10.2535/ofaj.79.121

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Event date： 2024.10

Language：English   Presentation type：Oral presentation (general)  

Venue：大連理工大学   Country：China  

Event date： 2023.10

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：酪農学園大学   Country：Japan  

Event date： 2023.9

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Venue：帯広畜産大学   Country：Japan  

Event date： 2023.9

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Venue：帯広畜産大学   Country：Japan  

Event date： 2023.6

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Venue：神奈川県相模原市（オンライン開催）   Country：Japan  

Event date： 2023.2

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Venue：Indonesia   Country：Indonesia  

Event date： 2022.10

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Venue：香川県高松市   Country：Japan  

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透けてきたSkeleton構造-骨格筋・結合組織の形態形成機構とその利用- コンドロイチン硫酸を通して明らかとなった筋管の形成

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Expression and intracellular localization of TBC1D9, a Rab GTPase-accelerating protein, in mouse testes
©2015 Japanese Association for Laboratory Animal Science. Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.

Event date： 2015.10

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©2015 Japanese Association for Laboratory Animal Science. Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.

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Event date： 2014.11

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細胞形態ダイナミクスをリードする分子群を求めて コンドロイチン硫酸 耳かき1杯の糖が決定する骨の運命

Event date： 2014.8

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ヒトコブラクダにおける網膜神経節細胞トポグラフィーと視力 上方向からの再現(Retinal Ganglion Cell Topography and Visual Acuity in Dromedary Camel: Re-Scene From Above)

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成熟ラット精巣における新規スフィンゴシン1リン酸受容体モチーフ蛋白質遺伝子の発現(Expression of a Novel Sphingosine 1-Phosphate Receptor Motif Protein Gene in Maturing Rat Testes)

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ウシ血リンパ節におけるHEVマーカの異なった差次的発現(Differential expression of HEV marker in bovine hemal node)

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獣医学領域における化学遺伝学の最前線 コンドロイチン硫酸の糖鎖構造が決定する細胞分化の方向性

Event date： 2012.3

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ウシ血リンパ節における血液の供給と微小血管系に関する研究(Studies on the blood supply and microvasculature system in bovine hemal node)

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獣医学領域における化学遺伝学の黎明 糖鎖を修飾する硫酸基の機能～コンドロイチン硫酸種の脂肪分化への作用

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ラット精巣におけるガン・精巣(CT)抗原遺伝子の発現解析(Expression analysis of CT (cancer/testis) antigen gene in maturing rat testis)

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Event date： 2010.9

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マウス網膜と視神経の形成におけるコンドロイチン硫酸プロテオグリカンの時空間的分布(Spatiotemporal distribution of chondroitin sulfate proteoglycans in the developing mouse retina and optic nerve)

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Event date： 2010.8

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牛に飼料とともに鮭皮由来コラーゲンペプチド（MCP）を8週間摂取させ、前肢蹄冠の背側、外側、指間の真皮を解析した。MCP群の背側と外側の蹄冠皮膚は真皮の厚さ、線維芽細胞数、コラーゲン細線維の直径が増加、上昇した。MCP群の指間真皮の線維芽細胞数は増加したが、厚さは低下し、コラーゲン細線維直径は減少した。線維径を調整するデルマタン硫酸とV型コラーゲンはMCP群のすべての部位で増加し、特に指間での増加が顕著であった。結合組織の強さの指数MADは背側と外側で増加したが指間では減少した。MCP投与による指間真皮のユニークな応答は細いコラーゲン細線維を集めて柔軟な結合組織を形成し、着地時の衝撃を緩衝するためと考察した。MCPは線維芽細胞に作用し蹄冠の各部位に即した太さのコラーゲン細線維を産生させ、真皮構造を強化する効果があると考えられた。

Event date： 2010.1

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牛に飼料とともに鮭皮由来コラーゲンペプチド（MCP）を8週間摂取させ、前肢蹄冠の背側、外側、指間の真皮を解析した。MCP群の背側と外側の蹄冠皮膚は真皮の厚さ、線維芽細胞数、コラーゲン細線維の直径が増加、上昇した。MCP群の指間真皮の線維芽細胞数は増加したが、厚さは低下し、コラーゲン細線維直径は減少した。線維径を調整するデルマタン硫酸とV型コラーゲンはMCP群のすべての部位で増加し、特に指間での増加が顕著であった。結合組織の強さの指数MADは背側と外側で増加したが指間では減少した。MCP投与による指間真皮のユニークな応答は細いコラーゲン細線維を集めて柔軟な結合組織を形成し、着地時の衝撃を緩衝するためと考察した。MCPは線維芽細胞に作用し蹄冠の各部位に即した太さのコラーゲン細線維を産生させ、真皮構造を強化する効果があると考えられた。

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マウスの胚は、妊娠4日目（膣栓確認日＝妊娠1日目）に子宮上皮に着床し、子宮内膜の脱落膜化、子宮管腔の閉塞を伴って胎盤形成期へと移行する。胚着床に必須な分子の一つ、白血病阻止因子（LIF）は、胚が子宮上皮へ接着した後の妊娠の進行に必須であることから、我々はLIFが制御する分子機構について研究を進めてきた。これまでの我々の研究で、LIFは、神経筋接合部で筋肉の収縮・弛緩制御に必須とされる、アグリン‐アセチルコリン受容体系を制御していることが明らかとなった。実際、胚が子宮上皮に接着した妊娠5日目には、アグリンは子宮上皮の頂面に移動することから、アグリンが子宮管腔の閉塞に関与すると予想した。そこで、本研究では、抗アグリン抗体を用いて機能阻害を試み、LIFの制御下にあるアグリンが、着床にどのように関与するのかについて検討を行った。ウサギを免疫動物として、LIF及びアグリンに対する抗体（protein A精製）を作製した。妊娠3日目のマウス（C57BL/6J及びICR）に、作製した抗体及びコントロールとして正常ウサギIgGを、計3回（妊娠3日目の12時、22時、妊娠4日目の10時）、1回当たり7.5 &micro;g/マウス重量（g）の濃度で、腹腔内に投与した。そして妊娠7日目に採材し、胚着床の進行について形態学的な検討を行った。正常ウサギIgGの投与では胚着床に変化は認められなかったが、抗LIF抗体では、C57BL/6Jマウスで完全に着床を阻害し、ICRマウスでも有意に着床数が減少した。採材した子宮からは着床を完了していない胚盤胞を回収した。このことから、腹腔内に投与した抗体は、子宮内で作用していることが示された。抗アグリン抗体の投与では、胚の着床数に有意な変化は認められなかったが、いくつかのマウスでは着床数の異常を伴って、脱落膜化の亢進や異常が認められた。以上より、アグリンは脱落膜細胞の増殖・分化を制御することにより、子宮管腔の閉塞を含むマウスの胚着床の過程に重要な役割を持つことが示唆された。

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Event date： 2009.2

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Antivascular photodynamic therapy (PDT) Suppresses tumor growth and prolonged the survival in solid tumor-bearing mice. The purpose of this study was to assess the efficacy of antivascular PDT using BPD-MA for treatment of oral and nasal tumors in 14 dogs. At 15 min after initiating intravenous infusion of 0.5 mg/kg benzoporphyrin derivative monoacid ring A, tumors were irradiated with laser light at 690 nm emitted by a diode laser, The 1-year survival rate of 7 dogs with oral tumors was 71%. The 1-year Survival rate of 7 does with nasal tumors was 57%. Imaging of each tumor was performed by using angiographic computed tomography before and after each antivascular PDT. Contrast-enhanced turners were observed before antivascular PDT, but these tumors were not enhanced with contrast medium following antivascular PDT. Antivascular PDT is suggested to be a promising method for dogs with oral and nasal tumors that cannot be effectively treated with current antitumor therapies.

Event date： 2009.2

Language：English  

Antivascular photodynamic therapy (PDT) Suppresses tumor growth and prolonged the survival in solid tumor-bearing mice. The purpose of this study was to assess the efficacy of antivascular PDT using BPD-MA for treatment of oral and nasal tumors in 14 dogs. At 15 min after initiating intravenous infusion of 0.5 mg/kg benzoporphyrin derivative monoacid ring A, tumors were irradiated with laser light at 690 nm emitted by a diode laser, The 1-year survival rate of 7 dogs with oral tumors was 71%. The 1-year Survival rate of 7 does with nasal tumors was 57%. Imaging of each tumor was performed by using angiographic computed tomography before and after each antivascular PDT. Contrast-enhanced turners were observed before antivascular PDT, but these tumors were not enhanced with contrast medium following antivascular PDT. Antivascular PDT is suggested to be a promising method for dogs with oral and nasal tumors that cannot be effectively treated with current antitumor therapies.

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Event date： 2009

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Language：Japanese  

透けてきたSkeleton構造-骨格筋・結合組織の形態形成機構とその利用- コンドロイチン硫酸を通して明らかとなった筋管の形成

Language：English  

Expression and intracellular localization of TBC1D9, a Rab GTPase-accelerating protein, in mouse testes
©2015 Japanese Association for Laboratory Animal Science. Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.

DOI： 10.1538/expanim.15-0016

Language：Japanese  

細胞形態ダイナミクスをリードする分子群を求めて コンドロイチン硫酸 耳かき1杯の糖が決定する骨の運命

Language：English  

ヒトコブラクダにおける網膜神経節細胞トポグラフィーと視力 上方向からの再現(Retinal Ganglion Cell Topography and Visual Acuity in Dromedary Camel: Re-Scene From Above)

Language：English  

成熟ラット精巣における新規スフィンゴシン1リン酸受容体モチーフ蛋白質遺伝子の発現(Expression of a Novel Sphingosine 1-Phosphate Receptor Motif Protein Gene in Maturing Rat Testes)

Language：English  

ウシ血リンパ節におけるHEVマーカの異なった差次的発現(Differential expression of HEV marker in bovine hemal node)

Language：English  

ウシ血リンパ節における血液の供給と微小血管系に関する研究(Studies on the blood supply and microvasculature system in bovine hemal node)

Language：Japanese  

獣医学領域における化学遺伝学の最前線 コンドロイチン硫酸の糖鎖構造が決定する細胞分化の方向性

Language：Japanese  

獣医学領域における化学遺伝学の黎明 糖鎖を修飾する硫酸基の機能～コンドロイチン硫酸種の脂肪分化への作用

Language：English  

ラット精巣におけるガン・精巣(CT)抗原遺伝子の発現解析(Expression analysis of CT (cancer/testis) antigen gene in maturing rat testis)

Language：English  

マウス網膜と視神経の形成におけるコンドロイチン硫酸プロテオグリカンの時空間的分布(Spatiotemporal distribution of chondroitin sulfate proteoglycans in the developing mouse retina and optic nerve)

Language：English  

DOI： 10.1016/j.tice.2010.08.001

Language：Japanese  

ウマにおいて疝痛を伴った内毒素血症はしばしば発症する疾病であって、毒素によって腸間膜動脈には様々な程度の虚血、動脈の痙攣や壊死が引き起こされる。重篤な場合では、それらの臨床症状が疝痛の前駆的発症要因になることが報告されている。エンドトキシンが関与し疝痛を患ったウマでは、腸間膜の血管における障害はこれまでのところ大いに注目されてきている。そこで、この研究では内毒素血症を自然発症したサラブレットにおいて、腸間膜動脈の障害を形態学的に検索した。この研究で得られた結果は、腸間膜にみられる血管の組織構造に対するエンドトキシンの効果をより良く理解できるものと思われる。試料は3.0％グルタルアルデヒド溶液に固定後、Quetol 812に包埋した。包埋した電顕試料より厚切り切片および超薄切片を作製し、前者には1％トルイジンブルー染色を施した後、光学顕微鏡を用いて、後者には酢酸ウラニウムとクエン酸鉛による二重染色を行った後、透過型電子顕微鏡にて詳細に検索した。発症群では動脈壁において血管内皮細胞の剥離、出血像、歪な形を呈した中膜平滑筋細胞や融解壊死した顆粒球などの血液細胞、空胞などが多数観察された。これらの結果は、すでに報告されているウマの内毒素血症の組織像と類似しており、また、エンドトキシンにより腸間膜中の動脈が損傷を受け、ウマの腸管において臓器性Shwartzman反応が起こったものと示唆された。

Language：Japanese  

角膜実質の再生医療 ブタ角膜実質細胞の培養法の検討

Language：Japanese  

ノックダウン細胞を用いた、デコリンとコラーゲン細線維の相互関連の解析

Language：English  

Active Expression of Matrix Metalloproteinase-13 mRNA in the Granulation Tissue of Equine Superficial Digital Flexor Tendinitis
The DNA microarray analysis for matrix metalloproteinase (MMP)-related mRNA expression in equine superficial digital flexor tendinitis indicated that mRNA level of MMP-13 was apparently up-regulated in the tendinitis as compared to normal tendon. In situ hybridization also revealed that fibroblastic cells proliferated in the granulation tissue generated in the tendinitis actively expressed MMP-13 mRNA. On the other hand, in normal tendon, a few fibroblastic cells and vascular components lied in the endotenon barely expressed its mRNA, but other cellular components in the tendon bundle were not positively hybridized. As mentioned above, MMP-13 but not other collagenases or gelatinases, may play an important role in tendon injuries in the racehorses.

Language：English  

Active expression of matrix metalloproteinase-13 mRNA in the granulation tissue of equine superficial digital flexor tendinitis
The DNA microarray analysis for matrix metalloproteinase (MMP)-related mRNA expression in equine superficial digital flexor tendinitis indicated that mRNA level of MMP-13 was apparently up-regulated in the tendinitis as compared to normal tendon. In situ hybridization also revealed that fibroblastic cells proliferated in the granulation tissue generated in the tendinitis actively expressed MMP-13 mRNA. On the other hand, in normal tendon, a few fibroblastic cells and vascular components lied in the endotenon barely expressed its mRNA, but other cellular components in the tendon bundle were not positively hybridized. As mentioned above, MMP-13 but not other collagenases or gelatinases, may play an important role in tendon injuries in the racehorses.

DOI： 10.1292/jvms.69.637

Language：Japanese  

皮膚真皮のコラーゲン線維とグリコサミノグリカンに対するコラーゲンペプチド摂取の効果

Language：Japanese  

Language：Japanese  

Language：English  

Differences in Tumor Necrosis Factor (TNF)α and TNF Receptor-1-Mediated Intracellular Signaling Factors in Normal, Inflamed and Scar-Formed Horse Tendons
Tumor necrosis factor (TNF) receptors (TNF-R)-mediated cell survival or apoptosis has been demonstrated in many cells, but little is known about survival or apoptotic signals via TNF-R1 in tendinocytes. In this study, we focused on four signaling factors, TNF&alpha;, TNF-R1, TNFR-associated factor2 (TRAF2) and caspase-3, in order to elucidate the signaling events in tendinocytes. Samples were obtained from normal, inflamed and scar-formed equine superficial digital flexor tendons. To detect these signaling factors, samples were subjected to immunohistochemistry and Western blot analysis, and some samples were also subjected to reverse transcription-polymerase chain reaction (RT-PCR), PCR-Southern blot analysis and in situ hybridization to detect the expression of TNF&alpha; mRNA. Distribution of the four factors differed depending on the tendon condition, normal, inflamed or scar-formed. In the normal tendon, large amounts of TRAF2 were found in tendinocytes, but the amounts of TNF-R1 were small. TNF&alpha; mRNA was expressed most highly in the inflamed tendon. TNF-R1, which was only faintly detected in the normal tendon, was detected at a high level in the inflamed tendon, and the amounts of TRAF2 and caspase-3 also increased. Activated caspase-3 was only detected in the inflamed tendon. TNF&alpha; mRNA was also expressed in the scar-formed tendon, though it showed weak signals, and the expression levels of TNF-R1, TRAF2 and caspase-3 proteins were very low. Two distinct intracellular signaling pathways of TNF&alpha;, which lead to cell survival and apoptosis, might be present in tendinocytes mediated through TNF-R1. These results, which reflect the dynamism of TNF&alpha;, provide important clues for means to prevent tendinopathy.

DOI： 10.1292/jvms.67.985

Language：English  

Control of the collagen fibril diameter in the equine superficial digital flexor tendon in horses by decorin
The distribution pattern of collagen fibril diameter in the equine superficial digital flexor tendon (SDFT) is known to differ in central and peripheral areas of some regions. This study reports the essence of collagen fibril differences among different regions of the equine SDFT by transmission electron microscopic (TEM) and high-voltage electron microscopic observations and biochemical analysis. The distribution of large collagen fibrils increased but the density of collagen fibrils decreased from the proximal metacarpal region to the distal metacarpal region. Large collagen fibrils with an irregular cross-sectional profile were found more frequently in the middle metacarpal region than in other regions. Three-dimensional reconstruction of images of irregularly shaped collagen fibrils revealed that these fibrils are formed through fusion of small collagen fibrils with large ones. The amount of decorin, which reportedly inhibits the lateral fusion of collagen fibrils, decreased in the direction of the distal metacarpal region. On the other hand, the size of decorin gradually increased in the direction of the distal metacarpal region. These results suggest that regional differences in collagen fibril distribution and density of collagen fibrils in the SDFT are due, at least in part, to fusion of collagen fibrils and the concomitant regional differences in the amount and size of decorin.

Language：English  

Control of the Collagen Fibril Diameter in the Equine Superficial Digital Flexor Tendon in Horses by Decorin
The distribution pattern of collagen fibril diameter in the equine superficial digital flexor tendon (SDFT) is known to differ in central and peripheral areas of some regions. This study reports the essence of collagen fibril differences among different regions of the equine SDFT by transmission electron microscopic (TEM) and high-voltage electron microscopic observations and biochemical analysis. The distribution of large collagen fibrils increased but the density of collagen fibrils decreased from the proximal metacarpal region to the distal metacarpal region. Large collagen fibrils with an irregular cross-sectional profile were found more frequently in the middle metacarpal region than in other regions. Three-dimensional reconstruction of images of irregularly shaped collagen fibrils revealed that these fibrils are formed through fusion of small collagen fibrils with large ones. The amount of decorin, which reportedly inhibits the lateral fusion of collagen fibrils, decreased in the direction of the distal metacarpal region. On the other hand, the size of decorin gradually increased in the direction of the distal metacarpal region. These results suggest that regional differences in collagen fibril distribution and density of collagen fibrils in the SDFT are due, at least in part, to fusion of collagen fibrils and the concomitant regional differences in the amount and size of decorin.

DOI： 10.1292/jvms.67.855

Language：Japanese  

若齢期のウマにおける伸筋腱および屈筋腱の組織学的変化

Language：Japanese  

ウマ浅指屈筋腱のコラーゲン細線維直径を変化させる分子要因について

Language：English  

Matrix metalloproteinase inhibitor RECK expression in canine tumors
Matrix metalloproteinases (MMPs) selectively degrade the extracellular matrix, and they have been reported to play an important role in tumor invasion, metastasis and angiogenesis. These enzymes are closely related to tumor malignancy and patient survival time. Recently, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene was identified as an endogenous membrane-anchored MMP inhibitor. The down-regulation of RECK has been implicated in tumor progression. In this study, the expression levels of the RECK messenger ribonucleic acid (mRNA) in various spontaneously developed canine tumors were investigated by using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and the correlation between RECK and clinicopathological factors, as well as MMP-9 expression were analyzed. The median age of 36 dogs investigated in this study was 9 years old (range, 1-15 years old). Quantitative RT-PCR could detect low levels of expression of RECK mRNA in the tumor samples. The expression levels of RECK mRNA in some tumor tissue samples were significantly lower than those in normal tissue samples. No significant associations of RECK with clinicopathological factors were observed. Using the Mann-Whitney U test, the expression level of the MMP-9 mRNA was observed to be significantly correlated to RECK expression (p&lt
0.05).

DOI： 10.1292/jvms.67.761

Language：Japanese  

腱変性過程での熱,炎症性サイトカインおよびゼラチナーゼの関連

Language：English  

Molecular cloning of canine membrane-anchored inhibitor of matrix metalloproteinase, RECK
The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5&#37; and 91.9&#37; homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation. migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.

Language：English  

Molecular Cloning of Canine Membrane-Anchored Inhibitor of Matrix Metalloproteinase, RECK
The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5&#37; and 91.9&#37; homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation, migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.

DOI： 10.1292/jvms.67.385

Language：Japanese  

馬の腱の損傷程度を反映する血清中I型コラーゲン架橋N-末端テロペプチド濃度

Language：Japanese  

ウマ浅指屈筋腱のコラーゲン細線維の形態は部位によって変化する

Language：Japanese  

膝関節の靱帯の構造特性

Language：Japanese  

The Molecular Marker NTx Reflecting Changes in Tissue Remodeling Associated with Injury to the Equine Superficial Digital Flexor Tendon
The aim of this study is to determine the usefulness of the serum level of type- I collagen cross-linked N-telopeptide (NTx)-originally a molecular marker of bone resorption-as a marker for detecting degradation of type-I collagen, especially in cases of equine tendonitis. NTx serum concentration, which was found to reflect tendon condition sensitively, was significantly higher in horses with tendonitis than in controls. Although its value was very high in the acute and subacute phases of tendonitis, the concentration gradually declined. In the chronic phase, there was no significant difference between NTx concentrations in horses with tendonitis and controls. This study revealed NTx to be potentially a valuable molecular marker for monitoring post-traumatic tendon condition. If an association between NTx concentration and tendinous degeneration can be established, it will become possible to identify horses with preclinical tendon injury easily.

DOI： 10.12935/jvma1951.58.175

Language：Japanese  

乳牛の胎盤停滞と子宮小丘の形態の関連性

Language：Japanese  

腱細胞のデコリン及びTGF-β1の産生はデコリン遺伝子の発現抑制によって制御される

Language：Japanese  

腱細胞へのデコリンアンチセンスオリゴヌクレオチド遺伝子の導入効果

Language：Japanese  

ウマ浅指屈筋腱におけるコラーゲン細線維の分岐

Language：English  

Focal fatty liver in a heifer: utility of ultrasonography in diagnosis.
A 30-month-old Holstein heifer presented with a history of decreased appetite and respiratory signs. Sonographic examination of the liver incidentally revealed an area of increased echogenicity between the portal vein and the gallbladder. The lesion was nonspherical and had no mass effect or displacement of the adjacent vessels. Its boundaries, to the liver, were geographic. The liver specimen was histologically compatible with a diagnosis of focal fatty liver change (FFLC). The sonographic features of focal fatty infiltration of the liver are characteristic. Recognition of the ultrasonographic data is important to differentiate FFLC from other lesions.

DOI： 10.1292/jvms.66.341

Language：English  

Focal fatty liver in a heifer: Utility of ultrasonography in diagnosis
A 30-month-old Holstein heifer presented with a history of decreased appetite and respiratory signs. Sonographic examination of the liver incidentally revealed an area of increased echogenicity between the portal vein and the gallbladder. The lesion was nonspherical and had no mass effect or displacement of the adjacent vessels. Its boundaries, to the liver, were geographic. The liver specimen was histologically compatible with a diagnosis of focal fatty liver change (FFLC). The sonographic features of focal fatty infiltration of the liver are characteristic. Recognition of the ultrasonographic data is important to differentiate FFLC from other lesions.

Language：English  

Focal fatty liver in a heifer: Utility of ultrasonography in diagnosis
A 30-month-old Holstein heifer presented with a history of decreased appetite and respiratory signs. Sonographic examination of the liver incidentally revealed an area of increased echogenicity between the portal vein and the gallbladder. The lesion was nonspherical and had no mass effect or displacement of the adjacent vessels. Its boundaries, to the liver, were geographic. The liver specimen was histologically compatible with a diagnosis of focal fatty liver change (FFLC). The sonographic features of focal fatty infiltration of the liver are characteristic. Recognition of the ultrasonographic data is important to differentiate FFLC from other lesions.

Language：Japanese  

腱組織の取込み能について

Language：Japanese  

イヌ膝関節における前十字靱帯と外側側副靱帯の構造的差異

Language：Japanese  

ウマ浅指屈筋腱と総指伸筋腱の形態学的および生化学的性状の比較

Language：Japanese  

ウマ浅指屈筋腱の部位差について

Language：Japanese  

ウマ浅指屈腱炎におけるマトリックスメタロプロテアーゼ活性

Language：Japanese  

馬浅指屈腱炎におけるマトリックスメタロプロテアーゼ活性

Language：Japanese  

犬の混濁を呈した角膜の形態学的及び生化学的解析

Language：Japanese  

犬膝関節十字靱帯,側副靱帯の力学的,形態学的及び生化学的相異

Language：Japanese  

血管壁に発現する陰性荷電について

Language：Japanese  

なぜウマの屈腱炎は骨腱付着部で発症しないのか?

Language：Japanese  

シロザケ(Oncorhynchus keta)卵表面の塩漬に対する形態学的変化

Language：Japanese  

淡水カメへのビタミンD3投与による鰓後体と上皮小体の応答

Language：Japanese  

腱細胞におけるTNFα細胞内シグナル伝達因子の発現

Language：Japanese  

腱炎における腫瘍壊死因子(TNF)αの関与

Language：Japanese  

ウマ浅指屈筋腱の加齢性変化に関する研究

Language：Japanese  

Age-related Changes in the Morphological and Biochemical Characteristics of the Equine Superficial Digital Flexor Tendon
Morphological and biochemical analyses of age-related changes in the superficial digital flexor tendon (SDFT) of thoroughbred horses showed a straightening of collagen-fibril orientation and a decrease in diameter with aging. Type III collagen content increased with aging. In terms of glycosaminoglcan composition, dermatan sulfate decreased and chondroitin sulfate increased. In the SDFT of thoroughbred horses, morphological age-related changes in collagen fibrils are accompanied by changes in biochemical composition.

Language：English  

Localization of cytokines in tendinocytes of the superficial digital flexor tendon in the horse
Although inflammatory activation of cytokines have been analyzed in various tissues, there have only been a few and as-yet-inconclusive studies on cytokines in equine tendons. In this study, the localizations of 4 cytokines (IL-1alpha, IL-1beta, TNFalpha and IFNgamma) in tendinocytes of the equine superficial digital flexor tendon (SDFT) were analyzed by the use of an immunohistochemical method. In inflamed tendons positive staining for all 4 cytokines antibodies were detected in endotedinieum cells and vascular epithelial cells. In contrast, negative or trace immunoreactions were obtained in many tendinocytes in the normal tendon. The variation in cellular immune responses depending on the kind of cytokine may reflect the physiological/pathological condition of the SDFT.

Language：English  

Localization of cytokines in tendinocytes of the superficial digital flexor tendon in the horse.
Although inflammatory activation of cytokines have been analyzed in various tissues, there have only been a few and as-yet-inconclusive studies on cytokines in equine tendons. In this study, the localizations of 4 cytokines (IL-1alpha, IL-1beta, TNFalpha and IFNgamma) in tendinocytes of the equine superficial digital flexor tendon (SDFT) were analyzed by the use of an immunohistochemical method. In inflamed tendons positive staining for all 4 cytokines antibodies were detected in endotedinieum cells and vascular epithelial cells. In contrast, negative or trace immunoreactions were obtained in many tendinocytes in the normal tendon. The variation in cellular immune responses depending on the kind of cytokine may reflect the physiological/pathological condition of the SDFT.

DOI： 10.1292/jvms.64.945

Language：English  

Localization of cytokines in tendinocytes of the superficial digital flexor tendon in the horse
Although inflammatory activation of cytokines have been analyzed in various tissues, there have only been a few and as-yet-inconclusive studies on cytokines in equine tendons. In this study, the localizations of 4 cytokines (IL-1alpha, IL-1beta, TNFalpha and IFNgamma) in tendinocytes of the equine superficial digital flexor tendon (SDFT) were analyzed by the use of an immunohistochemical method. In inflamed tendons positive staining for all 4 cytokines antibodies were detected in endotedinieum cells and vascular epithelial cells. In contrast, negative or trace immunoreactions were obtained in many tendinocytes in the normal tendon. The variation in cellular immune responses depending on the kind of cytokine may reflect the physiological/pathological condition of the SDFT.

Language：Japanese  

腱における炎症性サイトカインとアポトーシス関連シグナル伝達因子の発現

Language：Japanese  

ウマの浅指屈腱筋におけるゴルジ腱紡錘について

Language：Japanese  

ラットの膵臓B細胞分泌に対する酢酸投与の影響

Language：Japanese  

腱組織における炎症性サイトカインとアポトーシス発現の関連 ウマ浅指屈腱における解析

Language：Japanese  

ウマ腱細胞におけるアポトーシス発現とサイトカインの関与について

Language：Japanese  

イヌ子宮蓄膿症における子宮内膜の形態学的及び生化学的性状