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.     

Histochemistry of complex carbohydrates in the horse duodenal gland

Complex carbohydrates were examined in glandular cells of the horse duodenal gland by using lectin histochemical techniques. In the horse, the duodenal gland was distributed in the area from the uppermost part of the small intestine to a point about 6m caudal to the pylorus. It consisted of two types of cells, mucous and serous cells. The former was found in glands distributed almost all over this part, but the latter was present in glands distributed restrictedly to the uppermost part of the small intestine at a point about 10 cm caudal to the pylorus. The cytoplasm of the mucous cell contained neutral glycoproteins with different saccharide residues as alpha-D-mannose, N-acetyl-beta (1----4)-D-glucosamine, galactose, alpha-galactose, alpha-N-acetylglucosamine, beta-D-Gal (1----3)-D-GalNAc, alpha-L-fucose and sialic acid. On the other hand, the serous cell contained neutral and acid glycoproteins with different residues such as alpha-D-mannose, alpha-D-glucose, beta-D-galactose(1----3)-D-N-acetylgalactose, alpha-L-fucose, N-acetyl-alpha-D-galactosamine, N-acetyl-beta (1----4)-D-glucosamine, galactose, alpha-galactose, alpha-N-acetylglucosamine and sialic acid. It is also elucidated in the present study that lipase, an enzyme for digestion, is contained in the serous cell of the equine duodenal gland.     

Distribution of TNF receptors and TNF receptor-associated intracellular signaling factors on equine tendinocytes in vitro

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.     

Metabolic profiles and bile acid extraction rate in the liver of cows with fasting-induced hepatic lipidosis

This study was designed to monitor lipid profile in the portal and hepatic blood of cows with fasting-induced hepatic lipidosis, and to compare the results with those in the jugular blood. The work was also carried out to investigate bile acid (BA) in these vessels, and further to investigate BA extraction rate in the liver. Five cows were equipped with catheters in the portal, hepatic and jugular veins (day 0), fasted for 4 days (day 1-day 4) and then refed (day 5-day 11). Before morning feeding, blood was sampled before, during and after fasting from the catheterized vessels. In the portal blood, the concentration of non-esterified fatty acids (NEFA) showed a progressive increase and at day 5 there was an approximate twofold rise. Increased NEFA concentrations were also found similarly in the other two veins. At day 5, beta-hydroxybutyrate (BHBA) in the portal, hepatic and jugular blood rose to 197, 190 and 186% of the pre-fasting value, respectively. However, the concentrations of NEFA and BHBA in the three veins gradually returned to pre-fasting concentration during the refeeding period. Compared with the pre-fasting value at day 0, the content of liver triglyceride (TG) increased significantly at day 5 (P < 0.01). In the liver, the hepatic extraction rate of BA dropped from 3.1 times pre-fasting to 2.2 times during fasting. There were no significant differences in the concentrations of glucose, TG, total cholesterol, cholesterol esters, free cholesterol and phospholipids. The results of the current study show that metabolic alterations occur in the portal, hepatic and jugular veins during induction of hepatic lipidosis in cows, and mostly metabolites, with exception of BA concentration, run parallel. The decreased BA extraction rate in the liver of fasted cows was considered to reflect hepatic cell impairment caused by TG accumulation. Hopefully, the findings, at least in part, contribute to the explanation of the pathophysiology of hepatic lipidosis in dairy cows.     

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.     

Electron microscopic cytochemical studies of anionic sites in the rat spleen

The distribution patterns of the intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of endothelial cells (ECs) in blood vessels and reticular cells (RCs) in the splenic cord of the rat spleen were studied by an electron microscopic cytochemical method using polyethyleneimine (PEI) as a cationic probe. Spleens from adult male rats were perfusion-fixed with 0.5% glutaraldehyde -4% paraformaldehyde containing 0.05% cetylpyridinium chloride and then perfused with 0.5% PEI at pH 7.4. On the free surfaces (glycocalyx of the plasma membrane) of the ECs examined, distinct PEI-positive reactions were observed in blood vessels, such as trabecular arteries, central arteries, arterial capillaries, pulp veins and trabecular veins. These PEI-positive electron-dense substances in the trabecular arteries, central arteries, and trabecular veins took the shape of a band of 170-250 nm in thickness. On the other hand, the corresponding ultrastructure of the ECs lining the splenic sinuses and the RCs in the splenic cord showed exceedingly weak PEI reactions. The PEI-reactive deposits were significantly thinner than those in the above blood vessels. As the thickness of the electron-dense substances can be related to the density of the negative charge, these results suggest that there is a high intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of ECs in blood vessels where blood cells and plasma pass into the red pulp or are discharged from the red pulp. In contrast, the splenic sinuses and RCs, which are the main components of the red pulp, contain weakly negative-charged sites. This may contribute to the microcirculation of the splenic blood vessels and elucidate the possible physiological functions of the spleen, such as blood storage.     

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.     

An investigation of the relationship between duct system and A cell-rich and PP cell-rich pancreatic islets in the canine pancreas.

The pancreata of four six-month-old dogs of the same mother, two with both the pancreatic and accessory pancreatic ducts (X-type) and two with only the accessory pancreatic duct (Y-type), were examined in this study. To clarify the relationships between the type of pancreatic duct system and the composition of pancreatic endocrine cells, the pancreata were examined immunohistochemically using antiserum against four types of pancreatic hormones (glucagon, insulin, somatostatin and pancreatic polypeptide). In all areas of the X- and Y-type duct system pancreata, B cells accounted for 52-82% of the total number of islet cells, and D cells accounted for 4-15%. In the X-type ducts system, the percentages of A and PP cells in the right and left lobes of the pancreas differed greatly. It was found that A and PP cells appear in inverse proportion to each other and that there exist A cell-rich and PP cell-rich pancreatic islets. The A cell-rich pancreatic islets appeared in the left lobes along the accessory pancreatic duct, while the PP cell-rich pancreatic islets were observed in the right lobes along the pancreatic duct. The body of the pancreas contained both A cell-rich and PP cell-rich pancreatic islets. In the Y-type duct systems, A cell-rich pancreatic islets appeared in the right lobes. These findings indicate that the composition of A and PP cells in pancreatic islets is closely related to the type of duct system.     

Carbohydrate histochemistry of bovine duodenal glands.

The complex carbohydrates in the bovine duodenal glands were examined histochemically using 5 peroxidase-labeled lectins to achieve a better knowledge of their glycoprotein profiles. The presence of at least 2 different cell types and secretion of neutral mucin were observed in the bovine duodenal glands. The duodenal gland cells located in the central area of the lobules contained neutral glycoproteins with different saccharide residues as beta-D-gal-(1-3)-D-galNAc, alpha-D-galNAc, beta-(1-4)-D-glcNAc, NeuNAc and D-galNAc. The other cell types located in the peripheral area of the lobules contained alpha-L-Fuc, alpha-D-galNAc, beta-(1-4)-D-glcNAc, NeuNAc and D-galNAc. These findings seemed to be characteristic of a unique digestive process of the ruminant.     

Fine Structural and Histochemical Study of Equine Paneth Cells

Ultrastructure. lysozyme and glycoconjugate activity in duodenal Paneth cells were observed concurrently in the horse. Paneth cells were seen to uniformly line the base of the equine intestinal glands. The round secretory granules have centrally located electron densities with peripherally located electron lucent halos. Histochemically, the peripheral halo layer was positively stained for carbohydrates by the periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) method and the entire granules reacted positively to the WGA. The central core area reacted with anti-lysozyme. We identified a young (Type I) and an old (Type II) cell population in the same crypt, but we suggest that the observed populations are variations of the same cell type with the varied appearance due to aging of the secretory granules.