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Superficial digital flexor tendon (SDFT) of the bovine hindlimb originates from the caudolateral aspect of the distal femur and finally inserts onto the plantar aspect of the middle phalanges. In the present study, morphology and morphometry of the bovine SDFT at the muscle-tendon junction (MTJ), middle metatarsus (mM) and tendon-bone interface (TBI) were investigated. Cross-sectional morphology at the three regions of SDFT were oval, semioval and ring-formed, respectively. Significant difference in cross-sectional area was found only between MTJ-mM and mM-TBI (P

In horse, the characterizations of hyalocytes under the steady state are still unclear. Therefore, we investigated characterizations of hyalocytes in normal equine eyes by their immunohistochemical phenotype, histomorphology and distribution. Thirty-one eyes from 18 horses, divided into 4 groups (G) by age, were used: early (G1) and late gestation (G2) fetuses, 1- to 3-year-old (G3) and 8- to 24-year-old (G4) horses. Equine hyalocytes were histologically classified into 4 types, and they immunohistochemically expressed MHC II and CD163. Hyalocytes were detected on and/or around ciliary processes and pars plana in G2, G3 and G4, but were not located on retina and optic papilla. A significant increase in distribution was found between G2 and both G3 and G4, and the largest distribution was found at ciliary processes in these groups. Equine hyalocytes were characterized as residential ocular macrophage and MHC II antigen-bearing cell, accompanied by a pleomorphic appearance and located in the contiguous ciliary body. Our data provided characterizations of hyalocytes in normal equine eyes and may well contribute to improving the understanding of pathogenesis of equine ocular disease.

Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

Skeletal muscle is mainly composed of myofibers and intramuscular connective tissue. Bundles composed of many myofibers, with each myofiber sheathed in connective tissue called the endomysium, are packed in the perimysium, which occupies the vast bulk of the intramuscular connective tissue. The perimysium is a major determination factor for muscle texture. Some studies have reported that collagen peptide (Col-Pep) ingestion improves the connective tissue architecture, such as the tendon and dermis. The present study evaluated the effects of Col-Pep ingestion on the chicken iliotibialis lateralis (ITL) muscle. Chicks were allocated to three groups: the 0.15% or 0.3% Col-Pep groups and a control group. Col-Pep was administered by mixing in with commercial food. On day 49, the ITL muscles were analyzed by morphological observation and the textural property test. The width of the perimysium in the 0.3% Col-Pep group was significantly larger than other two groups. Although scanning electron microscopic observations did not reveal any differences in the architecture of the endomysium, elastic improvement of the ITL muscle was observed as suggested by an increase of the width of perimysium and improved rheological properties. Our results indicate that ingestion of Col-Pep improves the textural property of ITL muscle of chickens by changing structure of the perimysium.

In this study, we evaluated the effectiveness of isoprothiolane in preventing fatty liver in dairy cows. Eight pregnant Holstein cows with high body condition scores were studied. Four cows were administered isoprothiolane via feed at 10 g/day/head from 21 days before delivery (treated group), while the remaining four cows received no treatment (non-treated group). Liver samples from both groups were obtained 21 days before delivery and 2 days following delivery. No accumulation of lipid droplets in hepatocytes was observed in any cows prior to delivery. Following delivery, the accumulation of lipid droplets in hepatocytes was significantly reduced in the treated group compared to the non-treated group. The number of mitochondria per unit area was also significantly smaller in the treated group than in the non-treated group. Blood and biochemical test results from cows in the treated group showed an increase in mGOT levels and stable intrahepatic ATP production. These results suggest that isoprothiolane treatment intensified beta-oxidation in liver mitochondria, thereby activating the TCA cycle and maintaining intrahepatic ATP production.

Tendons are composed of collagen fibrils and proteoglycan predominantly consisting of decorin. Decorin is located on the d-band of collagen fibrils, and its glycosaminoglycan (GAG) chains have been observed between collagen fibrils with transmission electron microscopy. GAG chains have been proposed to interact with each other or with collagen fibrils, but its three-dimensional organization remains unclear. In this report, we used focused ion beam scanning electron microscopy to examine the three-dimensional organization of the GAG chain in the Achilles tendon of mature rats embedded in epoxy resin after staining with Cupromeronic blue, which specifically stains GAG chains. We used 250 serial back-scattered electron images of longitudinal sections with a 10-nm interval for reconstruction. Three-dimensional images revealed that GAG chains form a ring mesh-like structure with each ring surrounding a collagen fibril at the d-band and fusing with adjacent rings to form the planar network. This ring mesh model of GAG chains suggests that more than two GAG chains may interact with each other around collagen fibrils, which could provide new insights into the roles of GAG chains.

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.

Beneficial effects of collagen peptide ingestion to reduce serum triacylglycerides have been reported, suggesting that the physiological conditions of adipocytes are modulated following collagen peptide inges- tion. In this study, the effects of prolylhydroxyproline, a major digestive product of ingested collagen pep- tide in the blood, on the differentiation of mouse 3T3-L1 preadipocytes in vitro were investigated. 3T3-L1 preadipocytes were induced to differentiate to adipocytes and treated with prolylhydroxyproline, or with an amino acid mixture of proline and hydroxyproline as a control. The amount of lipid was not affected by these treatments. However, the size of the lipid droplet was significantly smaller when treated with prolylhydroxyproline compared to the amino acid mixture or the non-treated control. Proton-coupled oligopeptide transporters were expressed in non-differentiated and/or differentiated 3T3-L1 cells. These results suggest that prolylhydroxyproline might modulate the morphology of lipid droplets by incorpora- tion into adipocytes through the transporters.

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.

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 TNFα 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.

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 1 month after injection of collagenase. At 1 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-MMP-2 was similar to those in the normal group. These results suggest that COP injection promotes healing processes of the tendon injury.

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 microg/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 collagen fiber networks treated with siDT cells on days 3 and 7. In addition, after treatment with 5.0 or 25 microg/ml decorin, the l 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 leucine-rich repeat proteoglycan in the process of collagen fibril assembly.

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.