Optical imaging of mouse articular cartilage using the glycosaminoglycans binding property of fluorescent-labeled octaarginine.

OBJECTIVE: The aim of the current study was to examine the cartilage-specific binding property of polyarginine peptides (R4, 8, 12, and 16) and specifically to test octaarginine peptides for the optical imaging of articular cartilage in experimentally induced arthritis in mice. METHODS: Four rhodamine-labeled polyarginine peptides each with a different-length arginine chain (R4, 8, 12, or 16) were injected into the knee joints of C57BL/6J mice (n=20). The joints were excised 1h later and the fluorescent signal intensity in cartilage cryosections was compared for the four peptides. To examine the substrate of R8 in cartilage, femoral condyles obtained from another set of mice were treated with chondroitinase ABC (Ch'ase ABC), keratanase or heparitinase then immersed in R8-rhodamine. Fluorescent signals were examined by fluorescent microscopy. Next, R8-rhodamine was injected into the right knee joints of three control and three collagen antibody-induced arthritis (CAIA) mice, and fluorescent intensity in normal and degenerative cartilage was semi-quantitatively analysed on the histological sections using image software. Finally, femoral condyles from normal mice (n=2) and CAIA mice (n=2) were immersed in R8-rhodamine and calcein, then imaged using optical projection tomography (OPT). RESULTS: Fluorescent signals were specifically detected in the cartilage pericellular matrix from the surface to the tide mark but were completely absent in the calcified layer or bone marrow. The number of arginine residues significantly influenced peptide accumulation in articular cartilage, with R8 accumulating the most. The fluorescent signal in the femoral condylar cartilage diminished when it was treated with Ch'ase ABC. R8 accumulation was significantly decreased in the degenerative cartilage of CAIA mice, and this was demonstrated both histologically and in three-dimensional (3D)-reconstruction image by OPT. CONCLUSION: R8 may be a useful new experimental probe for optical imaging of normal and arthritic articular cartilage.

An effective combination of carbohydrases that enables reduction of dietary protein in broilers: importance of hemicellulase.

After observing the effects of purified cellulase (C), hemicellulase (H), pectinase (P), and their combinations on the in vitro digestibility of a corn-soybean meal broiler diet, we examined the associations between pectin breakdown and the digestibilities of CP and DM by using free galacturonic acid (GA) as an index of pectin breakdown. There was no significant effect of the single enzymes except for H. However, the enzyme combinations H + P, C + H, and C + H + P significantly increased CP and DM digestibilities, whereas the combination of C + P was not effective. Because H has activities of both H and P, these enzymes were considered to be important in stimulating digestion. Furthermore, when the enzymes increased CP and DM digestibilities, GA concentration was significantly higher, and clear correlations between CP and DM digestibilities and GA concentration were observed, whereas correlations between the digestibilities and concentration of glucose or xylose + mannose as indices of cellulose and hemicellulose breakdown, respectively, were not significant. From these observations, we hypothesized that a mixture of enzymes could increase the protein digestibility of broiler feed. Thus, in the in vivo experiment, low-protein (19% CP) diets made mainly of corn and soybean meal with or without mixed enzymes were prepared and given to broiler chicks. The birds given the diet containing mixed enzymes showed significantly higher BW gain, with higher CP and DM digestibilities than the birds given the diet without the mixed enzymes. Moreover, the growth rate was same as that of the birds given the normal (21% CP) diet. The results indicate that the mixed enzyme preparation can effectively degrade indigestible cell constituents and thus enable the protein of the broiler feed to become more digestible. Furthermore, the results indicate the importance of H as a rate-limiting factor of cell wall breakdown.

Trichostatin A, a histone deacetylase inhibitor, suppresses synovial inflammation and subsequent cartilage destruction in a collagen antibody-induced arthritis mouse model.

OBJECTIVE: To investigate the effect of the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), on joint inflammation and cartilage degeneration in a collagen antibody-induced arthritis (CAIA) mouse model. METHODS: CAIA mice were given daily subcutaneous injections of various concentrations of TSA (0, 0.5, 1.0, and 2.0 mg/kg) and various parameters were monitored for 14 days. On Day 15, the hind paws were examined histologically. To investigate the effects of TSA on the expressions of matrix metalloproteinase (MMP)-3, MMP-13, tissue inhibitor of MMP-1 (TIMP-1), and acetyl-H4 by chondrocytes, another group of mice was sacrificed on Day 6. In vitro direct effect of TSA was examined by real-time PCR for mRNA of type II collagen, aggrecan, MMP-3, and MMP-13 in murine chondrogenic ATDC5 cells after pro-inflammatory cytokine stimulation. RESULTS: In the TSA-treated group, clinical arthritis was significantly ameliorated in a dose-dependent manner. The severity of synovial inflammation and the cartilage destruction score were significantly lower in the TSA 2.0 mg/kg group compared to the other TSA-treated groups. On immunohistochemistry, the number of MMP-3 and MMP-13-positive chondrocytes was significantly lower in the TSA 2.0 mg/kg group than in the control group. In contrast, the number of TIMP-1-positive cells and acetyl-histone H4-positive cells was significantly higher in the TSA 2.0mg/kg group than in the control group. TSA suppressed interleukin 1-beta and tumor necrosis factor-alpha-stimulated up-regulation of MMP-3, but not MMP-13 mRNA expression by ATDC5. CONCLUSION: The systemic administration of TSA ameliorated synovial inflammation in CAIA mice. Subsequently cartilage destruction was also suppressed by TSA, at least in part, by modulating chondrocyte gene expression.

Novel transdermal photodynamic therapy using ATX-S10.Na(II) induces apoptosis of synovial fibroblasts and ameliorates collagen antibody-induced arthritis in mice.

We aimed to test the effect of transdermal photodynamic therapy (PDT) on synovial proliferation in vitro and in vivo, using a novel photosensitizer, ATX-S10.Na(II). Synovial fibroblasts were obtained from patients with RA (RASF). Cell viability with or without PDT was determined by MTT assay. Cell morphology was examined by light and transmission electron microscopy. DNA fragmentation was labeled by TUNEL stain. Collagen antibody-induced arthritis (CAIA) was induced in DBA/1 mice, and the effects of transdermal PDT were evaluated by clinical and histological examination. PDT showed drug concentration-dependent and laser dose-dependent cytotoxicity on RASF. TUNEL stain and TEM study revealed the induction of apoptotic cell death of RASF. Transdermal PDT significantly reduced clinical arthritis and synovial inflammation in this model of arthritis. These results suggest that transdermal PDT using ATX-S10.Na(II) might be a novel less invasive treatment strategy for small joint arthritis and tenosynovitis.

A mixture of pure cellulase, hemicellulase and pectinase improves broiler performance.

1. The effects of a mixture of pure enzymes (cellulase, hemicellulase and pectinase) and a commercial enzyme, Energex, were examined on performance and metabolisabilities in broiler chicks given a maize-soybean meal diet. Composition of the mixed enzyme was similar to Energex except that protease was not present. 2. Chicks were divided into three groups: control, mixed enzyme and Energex with 7 replicates per group. Male broiler chicks were raised at 25 degrees C in wire-floored cages for 12 d from 15 d of age. Feed and water were offered ad libitum. 3. The Energex group gained significantly more weight and the mixed enzyme group tended to gain more than the control. Feed intakes were similar and thus the feed conversion ratio of Energex was significantly improved while it tended to be improved by the mixed enzyme. 4. The mixed enzyme group showed significant improvement in carcase and muscle weight when compared with the control group. The mixed enzyme group also showed significant improvement in organic matter and crude protein metabolisabilities. In the groups given enzyme, abdominal fat weight tended to decrease. 5. It is concluded that a combination of cellulase, hemicellulase and pectinase is effective in improving organic matter and crude protein metabolisabilities and carcase yield of broilers on a maize-soybean meal diet.

Distribution of adrenomedullin-containing perivascular nerves in the rat mesenteric artery.

Distribution of adrenomedullin (AM)-containing perivascular nerve fibers was studied in rat mesenteric arteries. Many fibers containing AM-like immunoreactivity (LI) were observed in the adventitia. AM-LI fibers were abolished by cold storage denervation or capsaicin but not 6-hydroxydopamine. Double immunostainings showed colocalization of AM-LI with calcitonin gene-related peptide (CGRP)-LI. The dorsal root ganglia had many AM-positive cells and AM mRNA detected by RT-PCR. Electron microscopy study revealed high proportions of immunogold labeling for AM and colocalization of both AM-LI and CGRP-LI in unmyelinated nerve axons. These results suggest that AM-containing perivascular nerves are distributed in the rat mesenteric artery.

Resorption of the postero-superior corner of the lateral part of the mandibular condyle correlates with progressive TMJ internal derangement.

The purpose of this study was to test the hypothesis that resorption of postero-superior corner of lateral part of the mandibular condyle (RLC) occurs with the advancement of TMJ internal derangement (ID). One hundred and sixty-seven TMJs in 122 patients were studied. RLC was assessed on plane radiographs of frontal oblique projection. The correlation between the prevalence of RLC and radiological stage of ID was analysed and clinical correlations to the radiological stage of ID and the prevalence of RLC were discussed. The higher prevalence of RLC in joints with advanced ID was statistically significant (Mann-Whitney U-test, P< 0.05). There was no correlation between the prevalence of RLC and neither OA on the articular surface of the condyle nor clinical symptoms/signs. The result of this study suggests that RLC appears to occur in joints with advanced ID as a result of remodelling changes secondary to ID.

Relationship between increased horizontal condylar angle and resorption of the posterosuperior region of the lateral pole of the mandibular condyle in temporomandibular joint internal derangement.

OBJECTIVES: The purpose of this study was to analyse the relationship between horizontal condylar angle, temporomandibular joint (TMJ) disk displacement and resorption of the lateral pole of the mandibular condyle (RLC). METHODS: The study comprised 162 TMJs (81 patients). Disk displacement was assessed and the horizontal angle of the condyle was measured on axial MR images. RLC was assessed on oblique frontal projection plain radiographs. RESULTS: The horizontal angle was increased in joints with disk displacement without reduction (one-factor ANOVA and Scheffe's f-test, P<0.05). The angle was greater in joints with RLC than in the joints without RLC (Student's t-test, P<0.05). CONCLUSIONS: Results suggest that the horizontal condylar angle is increased in joints with disk displacement without reduction. A possible relationship exists between RLC and increased horizontal condylar angle.

Tea polyphenols reduce glucocorticoid-induced growth inhibition and oxidative stress in broiler chickens.

1. The effects of dietary polyphenols (PP) on growth and oxidative stress in the corticosterone (CTC) treated broiler chickens model were studied. 2. Chicks (Cobb strain) were divided into 3 (CTC) x 3 (PP) blocks and given diets containing CTC at concentrations of 0, 10 and 20 mg/kg. 3. The body weight gain was lower when the birds were treated with CTC. However, the high dose of PP tended to reduce the effect of CTC. 4. The abdominal fat content, plasma triglyceride concentration and liver weight were increased by CTC and reduced by PP. 5. Muscle and liver thiobarbituric acid reactive substance (TBARS) were elevated by CTC and these effects were reduced by PP. Plasma CTC concentration was increased by dietary CTC treatment and decreased by PP. 6. In conclusion, our results indicate that PP can minimise growth inhibition, hyperlipidemia and oxidative stress induced by CTC treatment in broiler chickens.

Alteration of the horizontal mandibular condyle size associated with temporomandibular joint internal derangement in adult females.

OBJECTIVES: The purpose of this retrospective study was to analyse the relationship between horizontal size of the mandibular condyle and internal derangement (ID) of the temporomandibular joint (TMJ). METHODS: One hundred and thirty-nine joints in 88 women aged over 18 years were included in this study. The horizontal condylar size was measured in the antero-posterior and medio-lateral (ML) dimensions using axial magnetic resonance (MR) images. Radiological findings of ID were also assessed from MR imaging. RESULTS: The condyles in the joints with permanent disk displacement were smaller than those in joints without displacement in both dimensions (Fisher's protected least significant difference, P<0.05). There were statistically significant correlations between horizontal condylar size in the ML dimension and both disk morphology and radiological stage of ID (Spearman's correlation coefficient by rank, P<0.05). CONCLUSIONS: The results of this study suggest a possible relationship between horizontal condylar size and disk displacement. It is also suggested that the condyle becomes smaller in the ML dimension with advancement of ID.

Ectopic osteogenesis with biphasic ceramics of hydroxyapatite and tricalcium phosphate in rabbits.

Porous calcium phosphate ceramics consisting of hydroxyapatite (HA) and tricalcium phosphate (TCP) with different HA to TCP ratio were implanted intramuscularly in rabbits for six months in order to carry out a comparative study on osteogenic activity of the ceramics. Bone formation was detected only in HT73 (HA to TCP ratio, 7-3) specimens. Other implants, HT28 (2-8) and HT010 (0-10), could not induce bone. After a six-month period of implantation, HT28 and HT010 implants showed obvious degradation of the implants changing their shape and size macro and microscopically. Microscopically, they showed aggregates of fine particles and appearance of multinucleated cells. However, HT73 implants was less degraded and could maintain their original structure macro and microscopically. This study showed that HT73 ceramics can induce bone in rabbit muscle tissue and it is considered that maintenance of porous structure, that is, degradation rate of the materials may be one of the affecting factors in ceramic-induced osteogenesis.

Repulsive charge mechanism serve to maintain lumens and cavities. An histochemical study of rat serosa and kidney.

The free surface of the rat peritoneum was covered with a rich negative-charged substance which is distinctly stained with cationic colloidal iron (pH 1.5). Neuraminidase digestion erased this iron stain. Treatment with Limax flavus agglutinin (LFA), which has specific affinity to sialic acid, interferred with iron staining on the serosal surface. Transmission electron microscopy of the peritoneal samples stained with colloidal iron showed that colloidal particles were deposited on the free surface of the mesothelial cells. At pH 1.5, the colloidal particles aggregated in a dotted fashion; in those stained at pH 7.0, the particles arranged in fine strands (100-300 nm in length). This difference may occur as a structural transformation due to pH level changes. The string like structure seemed to correspond well to membrane associated sialomucin. The urinary surface of the rat glomerular podocytes possessed negatively charged sites detectable with cationic colloidal iron even at pH 1.5. Neuraminidase and LFA treatments erased iron staining. Substance containing sialic acid such as podocalyxin on the podocyte surface may be stained. This study shows that negatively charged sites of the substance covering the free surface of these regions repulse each other to maintain the serosal cavities or the podocyte end-feet slits.

The extracellular matrix in the mature brain: its reactions to endo-alpha-N-acetylgalactosaminidase and collagenase.

The hippocampal subiculum in the adult rat brain contains many neurons with nerve cell surface glycoproteins which are linked by collagenous ligands to perineuronal proteoglycans. The nerve cell surface glycoproteins or their terminal N-acetylgalactosamines are digested by endo-alpha-N-acetylgalactosaminidase. The terminal N-acetylgalactosamines linked by the collagenous ligands are not digested by endo-alpha-N-acetylgalactosasminidase. The collagenous ligands associated with the terminal N-acetylgalactosamines were digested by collagenase. The newly exposed terminal N-acetylgalactosamines by this collagenase incubation were digested by endo-alpha-N-acetyl-galactosaminidase. These findings on the rat agree with those obtained in our previous studies of the adult mouse brain samples. Furthermore they emphasize our hypothetical model that the perineuronal proteoglycans are related--via collagen ligands--with the nerve cell surface glycoproteins.

The intensely positively charged perineuronal net in the adult rat brain, with special reference to its reactions to oxine, chondroitinase ABC, hyaluronidase and collagenase.

Light microscopic observations of healthy adult rat brain sections stained with anionic iron colloid indicated that 5-10% of neurons in the hippocampal subiculum and all neurons in the medial cerebellar nucleus possessed an intensely positively charged perineuronal net. This net was demonstrated to react to oxine, and therefore suggested to consist of guanidino compounds. It was further shown that the intensely positively charged perineuronal net, in accordance with the intensely negatively charged perineuronal net of proteoglycans, was digested by chondroitinase ABC, hyaluronidase, and collagenase, but not by endo-alphaN-acetylgalactosaminidase. This finding suggested that the former positively charged net might be linked to the latter negatively charged one.

Purkinje cells in the adult cat cerebellar cortex possess a perineuronal net of proteoglycans.

The Purkinje cells in the adult cat cerebellar cortex were found to possess perineuronal proteoglycans which could be stained with our fine cationic iron colloid and Fujita's highly concentrated aldehyde fuchsin, and digested by chondroitinase ABC/keratanase/ heparitinase and hyaluronidase. The Purkinje cells are surrounded by some collagenous elements which are stained with Gömöri's ammoniacal silver and digested by collagenase. The Purkinje cells also express nerve cell surface glycoproteins which are labeled with lectin Vicia villosa agglutinin and digested by a double treatment with collagenase and endo-alpha-N-acetylgalactosaminidase. Sole digestion by endo-alpha-N-acetylgalactosaminidase never erased the lectin labeling of the nerve cell surface glycoproteins. These findings suggest that the collagenous elements mediate the linkage of the perineuronal proteoglycans to the nerve cell surface glycoproteins. It is presumed that in mice and rats, the perineuronal nets of proteoglycans and nerve cell surface glycoproteins of the Purkinje cells are so thin or coarse that they can not be sufficiently visualized under the light microscope.

A study of the relationship between the position of the condylar head and displacement of the temporomandibular joint disk.

OBJECTIVES: To examine the possible relationship between condylar position and disk displacement in the TMJ. METHODS: Forty-eight joints with no disk displacement (NDD), 84 joints with a reducible displaced disk (RDD) and 99 joints with a permanently displaced disk (PDD) were selected. The positions of the condyle and TMJ disk were calculated from lateral MR images. RESULTS: There were significant differences in condylar position between the joints with NDD and RDD, while there was no difference between those with NDD and PDD (Kruskal-Wallis and post-hoc test, P<0.05). There was also a significant correlation between the position of the condylar and the displaced disk (Spearman's rank correlation, P<0.05). The condyle was located more anteriorly with advancing disk displacement. CONCLUSIONS: We suggest that if the disk displacement is slight, the condyle is displaced posteriorly: as the disk displacement becomes more severe (i.e. more anteriorly displaced), the condyle returns to the concentric position.

A study of factors for successful splint capture of anteriorly displaced temporomandibular joint disc with disc repositioning appliance.

The purpose of this study was to analyse the factors which influenced the success of disc recapture by the insertion of a disc repositioning appliance. Fifty-one joints with joint clicking that occurred at both middle to late opening and late closing (near maximum cuspation) of the mandible were splinted with a mandibular full-coverage repositioning appliance. The clinical and MR findings were compared between the joints with successful and unsuccessful splint disc capture. Thirty-two clicking joints with reducibly displaced discs (DDWR) had successful disc recapture, while six of 19 joints with displaced disc without reduction (DDWOR). Unsuccessful joints with DDWOR had significantly higher prevalence of deformed disc and joint effusion, higher VAS quantitative pain score, and severe disc displacement especially in medial part of the joint (P < 0.05). From the results of this study joints with DDWR can be expected to have successful disc recapture with the insertion of the appliance. In joints with DDWOR, presence of inflammatory conditions, changed disc morphology and extensive disc displacement in medial part of the joint are negative factors.

A high dose of vitamin E inhibits adrenal corticosterone synthesis in chickens treated with ACTH.

The present experiment was conducted to study the effects of dietary vitamin E on plasma corticosterone (CTC) concentration and adrenal steroid syntheses in chickens treated with adrenocorticotropic hormone (ACTH). Chickens were divided into ACTH(-) and ACTH(+) groups, and each group was further divided into three subgroups administered with vitamin E (500 or 5,000 mg/kg diet) and without the vitamin. Vitamin E (DL-alpha-tocopheryl acetate) was mixed with the basal diet at levels of 500 and 5,000 mg/kg and fed for 6 d. ACTH (20 IU/kg body weight) was given daily by intraperitoneal injection for 5 d. alpha-Tocopherol levels in the plasma and adrenal gland were markedly elevated by vitamin E feeding, and the level of adrenal free cholesterol (CHOL), which is used for steroid synthesis, was significantly decreased by vitamin E feeding in a dose-dependent manner. However, the level of adrenal CHOL ester was unchanged by any treatment. The elevations of pregnenolone, progesterone and CTC levels in the adrenal gland of chickens with ACTH treatment were decreased by vitamin E administration. The elevation of plasma CTC concentration in the ACTH(+) group was dramatically decreased by vitamin E administration, while that concentration was not influenced by the vitamin administration in the ACTH(-) group. These findings indicate that vitamin E suppresses the elevation of the plasma CTC concentration due to ACTH in chickens, possibly by inhibiting the conversion of CHOL ester to free CHOL in the adrenal gland.

Chewing ability as a parameter for evaluating the disability of patients with temporomandibular disorders.

Restoration of chewing ability is an important aspect of the treatment for temporomandibular disorders (TMDs). However, too little attention has been paid to it. We have used a questionnaire to evaluate and score the chewing ability of TMD patients. The questionnaire includes 19 kinds of food and a chewing task. The patient was asked if she/he experiences difficulty in enjoying eating. The aim of this study was to evaluate correlations between score of chewing ability (SCA) and other symptoms/signs of TMD. Four hundred and seventy-three consecutive TMD patients were evaluated for SCA and other symptoms/signs including temporomandibular joint (TMJ) pain, TMJ and muscle tenderness, TMJ noise (clicking and crepitus), and maximum mouth opening. The relationship between SCA and other symptoms/signs were analysed by multiple regression analysis. Score of chewing ability correlated significantly with TMJ pain and mouth opening capacity but not with TMJ noise and muscle tenderness. Age was a background factor but sex was not. The result of this study suggests that SCA correlated with dysfunction of the TMD patients. This method could be used to evaluate the ability of chewing in assessment of TMD.