Objective: To explore the pathological characteristics of three mice models of temporomandibular joint degenerative joint disease (TMJDJD), including osteoarthritis and osteoarthrosis, and to provide references for animal experimental study regarding the pathological mechanism of osteoarthritis and osteoarthrosis. Methods: A total of 54 8-week-old male C57BL/6 mice were selected to construct three TMJDJD animal models, including bilateral temporomandibular joint (TMJ) Freund's complete adjuvant (FCA) injection model, bilateral TMJ monosodium iodoacetate (MIA) injection model, and right TMJ discectomy model. FCA injection model (15 mice) was divided into saline injection group, FCA injection group-1 week, FCA injection group-2 week, FCA injection group-4 week and FCA injection group-6 week, 3 mice were used at each time point, with a total of 6 TMJs on both sides. MIA injection model (15 mice) was separated into saline injection group, MIA injection group-1 week, MIA injection group-2 week, MIA injection group-4 week and MIA injection group-6 week, 3 mice were used at each time point, with a total of 6 TMJs on both sides. TMJ discectomy model (24 mice) was split into control group, discectomy group-2 week group, discectomy group-4 week and discectomy group-6 week, six mice were used at each time point, with a total of six right TMJs. General pictures of the bilateral joints area of mice were collected 1 day after drug injection, and stereoscopic images of condylar tissues were collected 4 weeks after microsurgery for discectomy. Mouse TMJ tissue sections from each time point were stained with HE and toluidine blue, respectively, synovial tissues were scored for synovial inflammation, and the percentage of proteoglycan in condylar cartilage was quantitatively analyzed. Results: One day after intra-articular FCA or MIA injection, the width of bilateral TMJ were significantly increased in FCA injection groups [(24.60±0.46) mm] compared with the saline injection group [(21.63±0.52) mm] (t=4.25, P<0.013), the width of bilateral TMJ in MIA injection groups [(24.50±0.62) mm] were also significantly higher than that in saline injection group [(21.40±0.52) mm] (t=3.82, P=0.019). The synovitis scores in FCA injection groups 1, 2, 4, 6 weeks after FCA injection were significantly higher than that of the saline injection group (F=18.09, P<0.001), with the proteoglycan of condylar cartilage increased firstly and then decreased compared with the saline injection group (F=21.59, P<0.001). Condylar cartilage proteoglycan loss in different degrees were observed 1, 2, 4 and 6 weeks after MIA injection (F=13.59, P<0.001), and synovitis scores were increased at different degrees compared with saline injection group (F=14.79, P<0.001). The morphology of condylar cartilage in discectomy groups mice were severely damaged, synovial tissues showed dense connective tissue lesions at 2, 4 and 6 weeks postoperatively, condylar cartilage tissues showed a time-dependent loss of proteoglycan compared with the control group (F=40.62, P<0.001). Conclusions: Intra-articular FCA injection establishes a mouse model of TMJ osteoarthritis with severe synovial inflammation. Intra-articular MIA injection constructs a mouse model of typical TMJ osteoarthritis. Discectomy establishes a mouse TMJ osteoarthrosis model with severe condylar cartilage destruction.
Mice ; Male ; Animals ; Cartilage, Articular ; Osteoarthritis/pathology* ; Iodoacetic Acid ; Tolonium Chloride ; Mice, Inbred C57BL ; Temporomandibular Joint/pathology* ; Disease Models, Animal ; Proteoglycans ; Synovitis/pathology* ; Inflammation/pathology*

The rhizomes of Dioscorea japonica Thunb. are widely consumed as food and also used to treat diabetes and polyuria in Korea. This study was undertaken to study the anti-atopic dermatitis effects of a 95% ethanolic extract (DJE) of D. japonica in an oxazolone-stimulated murine model of atopic dermatitis (AD). The therapeutic effects of DJE on AD-like skin lesions were assessed on both ears. DJE (1%) or dexamethasone (0.5%; the positive control) were applied to skin lesions for three weeks. Serum levels of IgE and IL-4 were assessed by ELISA (enzyme-linked immunosorbent assay). Histopathological examinations were performed by hematoxylin and eosin (H&E) and toluidine blue staining and revealed DJE significantly reduced dermal thickness and inflammatory cell infiltration when applied to oxazolone-treated ear skin. DJE-treated AD mice also showed lower serum levels of IgE and IL-4 than oxazolone-stimulated controls. Our findings demonstrate DJE might be a useful safe, topical agent for the treatment of atopic diseases.
Animals ; Dermatitis ; Dermatitis, Atopic ; Dexamethasone ; Dioscorea ; Ear ; Enzyme-Linked Immunosorbent Assay ; Eosine Yellowish-(YS) ; Ethanol ; Hematoxylin ; Immunoglobulin E ; Interleukin-4 ; Korea ; Mice ; Oxazolone ; Polyuria ; Rhizome ; Skin ; Therapeutic Uses ; Tolonium Chloride

Charcot-Marie Tooth disease type 1A (CMT1A), the major type of CMT, is caused by duplication of peripheral myelin protein 22 (PMP22) gene whose overexpression causes structural and functional abnormalities in myelination. We investigated whether miRNA-mediated regulation of PMP22 expression could reduce the expression level of PMP22, thereby alleviating the demyelinating neuropathic phenotype of CMT1A. We found that several miRNAs were down-regulated in C22 mouse, a CMT1A mouse model. Among them, miR-381 could target 3′ untranslated region (3′UTR) of PMP22 in vitro based on Western botting and quantitative Real Time-PCR (qRT-PCR) results. In vivo efficacy of miR-381 was assessed by administration of LV-miR-381, an miR-381 expressing lentiviral vector, into the sciatic nerve of C22 mice by a single injection at postnatal day 6 (p6). Administration of LV-miR-381 reduced expression level of PMP22 along with elevated level of miR-381 in the sciatic nerve. Rotarod performance analysis revealed that locomotor coordination of LV-miR-381 administered C22 mice was significantly enhanced from 8 weeks post administration. Electrophysiologically, increased motor nerve conduction velocity was observed in treated mice. Histologically, toluidine blue staining and electron microscopy revealed that structural abnormalities of myelination were improved in sciatic nerves of LV-miR-381 treated mice. Therefore, delivery of miR-381 ameliorated the phenotype of peripheral neuropathy in CMT1A mouse model by down-regulating PMP22 expression. These data suggest that miRNA can be used as a potent therapeutic strategy to control diseases with copy number variations such as CMT1A.
Animals ; Demyelinating Diseases ; In Vitro Techniques ; Mice ; MicroRNAs ; Microscopy, Electron ; Myelin Sheath ; Neural Conduction ; Peripheral Nervous System Diseases ; Phenotype ; Sciatic Nerve ; Tolonium Chloride ; Tooth Diseases ; Untranslated Regions

OBJECTIVE: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. METHODS: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. RESULTS: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. CONCLUSION: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.
Avena ; Chromatin* ; Chromomycin A3 ; DNA Methylation* ; DNA Nucleotidylexotransferase ; DNA* ; Enzyme-Linked Immunosorbent Assay ; Humans ; Male ; Methylation ; Polymerase Chain Reaction ; RNA, Messenger* ; Semen ; Spermatozoa* ; Tolonium Chloride ; World Health Organization

OBJECTIVE: The aim of this study was to compare the efficacy of swim-up and density gradient centrifugation (DGC) for reducing the amount of sperm with fragmented DNA, sex chromosome aneuploidy, and abnormal chromatin structure. METHODS: Semen samples were obtained from 18 healthy male partners who attended infertility clinics for infertility investigations and were processed with swim-up and DGC. The percentages of sperm cells with fragmented DNA measured by the sperm chromatin dispersion test, normal sex chromosomes assessed by fluorescence in situ hybridization, and abnormal chromatin structure identified by toluidine blue staining were examined. RESULTS: The percentage of sperm cells with fragmented DNA was significantly lower in the swim-up fraction (9.7%, p=0.001) than in the unprocessed fraction (27.0%), but not in the DGC fraction (27.8%, p=0.098). The percentage of sperm cells with normal X or Y chromosomes was comparable in the three fractions. The percentage of sperm cells with abnormal chromatin structure significantly decreased after DGC (from 15.7% to 10.3%, p=0.002). The swim-up method also tended to reduce the percentage of sperm cells with abnormal chromatin structure, but the difference was not significant (from 15.7% to 11.6%, p=0.316). CONCLUSION: The swim-up method is superior for enriching genetically competent sperm.
Aneuploidy* ; Centrifugation, Density Gradient* ; Chromatin ; DNA Fragmentation* ; DNA* ; Fluorescence ; Humans ; In Situ Hybridization ; Infertility ; Male ; Methods ; Semen ; Sex Chromosomes* ; Spermatozoa* ; Tolonium Chloride ; Y Chromosome

OBJECTIVE: Diabetes mellitus (DM) is known to cause many systemic complications as well as male infertility. Astaxanthin (ASTX) is a powerful antioxidant that is involved in a variety of biologically active processes, including those with anti-diabetes effects. The present study investigates the effect of ASTX on the spermatozoa function in streptozotocin (STZ)-induced diabetic rats. METHODS: We divided 30 adult rats into three groups (10 rats per group), with a control group that received corn oil mixed with chow. DM was induced by intra-peritoneal injection of STZ. Eight weeks after the STZ injection, half of the diabetic animals were used as diabetic controls, and the rest were treated with ASTX for 56 days. Then the parameters and chromatin integrity of the epididymal sperm were analyzed using chromomycin A3, toluidine blue (TB), and acridine orange (AO) staining. RESULTS: The count, viability, and motility of the epididymal sperm were decreased significantly in the STZ group in comparison with the control group (count and viability, p<0.001; motility, p<0.001;0.01). ASTX increased normal morphology and viable spermatozoa compared to the STZ group (morphology, p=0.001; viability, p<0.001;0.05). The percentage of abnormal chromatins in TB and AO staining was higher in the STZ group compared to the control group (p<0.001;0.001). The mean percentage of TB and AO positive spermatozoa in STZ rats was significantly lower in the STZ+ASTX group (TB, p=0.001; AO, p<0.001;0.05). CONCLUSION: This study observed that in vivo ASTX treatment partially attenuates some detrimental effect of diabetes. Conversely, ASTX improved sperm viability, normal morphology, and DNA integrity.
Acridine Orange ; Adult ; Animals ; Chromatin ; Chromomycin A3 ; Corn Oil ; Diabetes Mellitus ; Dietary Supplements* ; DNA* ; Humans ; Infertility, Male ; Male ; Rats* ; Spermatozoa* ; Streptozocin ; Tolonium Chloride

PURPOSE: To evaluate the early histological effects of the intravesical instillation of platelet-rich plasma (PRP) in rabbit models of interstitial and hemorrhagic cystitis. METHODS: Thirty-six rabbits were classified into 6 groups: saline (S), S+PRP, hydrochloric acid (HCl), HCl+PRP, cyclophosphamide (CyP), and CyP+PRP. At 48 hours after induction, PRP was prepared and intravesically administered to the S+PRP, HCl+PRP, and CyP+PRP groups. Bladder sections were stained with toluidine blue for mast cell counting and with hematoxylin and eosin for histopathology and mitotic index determination. The proliferation index was determined by proliferating cell nuclear antigen (PCNA) immunolabeling. The nonparametric Mann-Whitney U-test was used for statistical analysis. RESULTS: No abnormalities were observed in the S group, whereas increased interstitial edema and increased average mitotic and proliferation indices were observed in the S+PRP group (P=0.023, P=0.004, and P=0.009, respectively). Intense epithelial loss, hemorrhage, and leukocyte infiltration were detected in the HCl and HCl+PRP groups, whereas a significantly increased average mitotic index was observed in the HCl+PRP group (P=0.002). When compared with its CyP counterpart, a significant reduction in hemorrhage and an increase in leukocyte infiltration and mitotic index were observed in the CyP+PRP group (P=0.006, P=0.038, and P=0.002, respectively). In addition, PCNA staining revealed a significantly increased proliferation index in the HCl+PRP and CyP+PRP groups (P=0.032 and P=0.015, respectively). CONCLUSIONS: The intravesical instillation of PRP increased the mitotic index in the saline and cyclophosphamide groups while decreasing macroscopic bleeding.
Administration, Intravesical* ; Cyclophosphamide ; Cystitis* ; Cystitis, Interstitial ; Edema ; Eosine Yellowish-(YS) ; Hematoxylin ; Hemorrhage ; Hydrochloric Acid ; Leukocytes ; Mast Cells ; Mitotic Index ; Platelet-Rich Plasma* ; Proliferating Cell Nuclear Antigen ; Rabbits ; Tolonium Chloride ; Urinary Bladder

BACKGROUND/AIMS: Previous studies have revealed that mast cells (MCs) may activate the protease-activated receptors and release of neuropeptides involved in the pathogenesis of irritable bowel syndrome (IBS). The levels of protease-activated receptor 2 (PAR-2) and tryptase can contribute to understanding the pathogenesis of IBS. METHODS: Colonoscopic biopsies were performed of 38 subjects (20 with IBS-diarrhea [IBS-D], eight with IBS-constipation [IBS-C], and 10 healthy volunteers). The mRNA and protein levels of tryptase and PAR-2 were assessed by real-time PCR and Western blot. The levels of vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene-related peptide (CGRP) were measured by immunohistochemistry, and MCs were counted by toluidine blue staining. RESULTS: Significant increases in the mRNA expression of tryptase (p<0.05, IBS-D, IBS-C vs control) and PAR-2 (p<0.05, IBS-D, IBS-C vs control) and in the tryptase protein level (p<0.05, IBS-D, IBS-C vs control) were detected in IBS. Elevations of MCs, CGRP, VIP and SP (p<0.05, IBS-D vs control) were observed for IBS-D only. CONCLUSIONS: Tryptase levels may upregulate the function of PAR-2, resulting in the release of neuropeptide and they were correlated with clinical symptoms associated with IBS.
Biopsy ; Blotting, Western ; Calcitonin Gene-Related Peptide ; Immunohistochemistry ; Inflammation ; Irritable Bowel Syndrome* ; Mast Cells ; Neuropeptides ; Real-Time Polymerase Chain Reaction ; Receptor, PAR-2* ; Receptors, Proteinase-Activated ; RNA, Messenger ; Substance P ; Tolonium Chloride ; Tryptases* ; Vasoactive Intestinal Peptide

BACKGROUND AND OBJECTIVES: Recurrent laryngeal nerve (RLN) damage commonly occurs from a thyroid surgery and causes communication impairment, aspiration and dysphagia. The purpose of this study is to develop a polycaprolactone (PCL) nerve guide conduit (NGC) coated with conductive materials for facilitating regeneration from the RLN defects and to evaluate the usefulness of the PCL NGC coated with conductive materials in a rabbit model. MATERIALS AND METHODS: The PCL NGCs coated with conductive materials were fabricated for this study. The types of conductive materials were single-walled carbon nanotubes (SWNTs) and poly (3,4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT:PSS) which were coated on the PCL NGCs by layer-by-layer (LBL) assembly techniques. An 8-mm segment of left RLN was resected in 24 New Zealand white rabbits. Three different NGCs (PCL and PCL with two conductive materials) were interposed between both stumps and fixed with suture. For the assessment of functional regeneration, the vocal cord mobility was observed using endoscopic system after RLN stimulation, and the motion change was analyzed. The atrophies of thyroarytenoid muscle and nerve growth were evaluated by Hematoxylin-Eosin (H-E) and toluidine blue (T-B) staining, respectively. Immunohistochemical study using anti-neurofilament, S-100 staining was further performed to evaluate the nerve regeneration. RESULTS: In endoscopic evaluation, the group with conductive PCL NGCs showed an improved tendency of vocal cord mobility compared to that of the other group. Nerve growth was observed with the time for 8 weeks in all groups and immunohistochemical staining revealed the expression of neurofilament and S-100 in regenerated nerve in all groups. The atrophies of thyroarytenoid muscle in the group with conductive PCL NGCs was also shown to be decreased compared to that of the nonconductive PCL NGC group. CONCLUSION: The study shows that PCL NGC coated with conductive materials appears to be a good alternative option for the repair and regeneration of RNL damages.
Atrophy ; Deglutition Disorders ; Laryngeal Muscles ; Nanotubes, Carbon ; Nerve Regeneration ; Polystyrenes ; Rabbits ; Recurrent Laryngeal Nerve* ; Regeneration* ; Sutures ; Thyroid Gland ; Tolonium Chloride ; Vocal Cords

Peripheral ameloblastoma (PA) in gingiva is rare and often confused with oral basal cell carcinoma (OBCC). The tissues of one case of PA and one case of OBCC with the same mandibular molar area affected were compared via an immunohistochemical examination using 50 antisera. The PA and OBCC showed similar proliferation of basaloid epithelial strands, but toluidine blue staining revealed that the PA had pinkish juxta-epithelial myxoid tissue, whereas the OBCC was infiltrated by many mast cells. Immunohistochemical comparisons showed that the PA was strongly positive for ameloblastin, KL1, p63, carcinoembryonic antigen, focal adhesion kinase, and cathepsin K, and slightly positive for amelogenin, Krox-25, E-cadherin, and PTCH1, whereas the OBCC was not. On the other hand, the OBCC was strongly positive for EpCam, matrix metalloprotease (MMP)-1, alpha1-antitrypsin, cytokeratin-7, p53, survivin, pAKT1, transforming growth factor-beta1, NRAS, TGase-1, and tumor nescrosis factor-alpha, and consistently positive for beta-catenin, MMP-2, cathepsin G, TGase-2, SOS-1, sonic hedgehog, and the beta-defensins-1, -2, -3, while the PA was not. These data suggest that the tumorigeneses of PA and OBCC differ, and that PAs undergo odontogenic differentiation and generate oncogenic signals for infiltrative growth and bone resorption, whereas OBCCs undergo basaloid epidermal differentiation as a result of growth factor/cytokine-related oncogenic signals.
Ameloblastoma* ; Amelogenin ; beta Catenin ; Bone Resorption ; Cadherins ; Carcinoembryonic Antigen ; Carcinogenesis ; Carcinoma, Basal Cell* ; Cathepsin G ; Cathepsin K ; Focal Adhesion Protein-Tyrosine Kinases ; Gingiva ; Hand ; Hedgehogs ; Immune Sera ; Immunohistochemistry ; Keratin-7 ; Mast Cells ; Molar* ; Tolonium Chloride