Histological evaluation of tissue destruction in mouse tongues caused by cryosurgery.

Introduction: Cryosurgery is recognized as a treatment option for various types of oral lesions. Although cryosurgery is less invasive and easier to perform than surgical treatments, adverse events, such as stomatitis and scarring can occur if the freezing is excessive. There are few studies regarding the effects of cryosurgery on the surrounding soft tissues. Thus, this study investigated the extent of tissue destruction and healing progress in tongues of mice who underwent cryosurgery. Methods: Eight-week-old male BALB/c mice were used. An instrument cooled with liquid nitrogen was lightly touched on the right side of the tongue for 5 s, and a second test was performed 10 s later. Histological evaluation was performed 3, 7, and 14 days after cryosurgery. Blood vessels were evaluated with India ink at 1, 3, 7, 14, and 21 days after cryosurgery. Results: Destruction of the soft tissue spread to the left side of the tongue after 3 days. At 7 days, it was confirmed that the muscle tissue was in the process of repair and was completely repaired at 14 days. Although blood vessels were not confirmed at 3 days, they were visible after seven days and were confirmed at 21 days all over the tongue. Discussion and Conclusion: These results indicated that the tissue destruction caused by cryosurgery was extensive and suggest that the duration and frequency of freezing should be minimized for clinical use. Lay Summary: Cryosurgery is a treatment method for various types of oral lesions. Freezing the lesion causes the tissue to collapse, resulting in its disappearance. Although cryosurgery is less invasive and easier to perform than surgical treatments, adverse events, such as stomatitis and scarring can occur if the freezing is excessive. This study investigated the extent of tissue destruction and healing progress in tongues of mice who underwent cryosurgery.The right side of mice tongues were frozen by an instrument cooled with liquid nitrogen for 5 s, and a second test was performed 10 s later. The tissue destruction was evaluated at 3, 7, and 14 days after freezing. Blood vessels were evaluated with India ink at 1, 3, 7, 14, and 21 days after freezing. Tissue destruction spread to the left side of the tongue after 3 days. At 7 days, it was confirmed that the muscle tissue was in the process of repair and was completely repaired at 14 days. Blood vessel repair was confirmed at 21 days in the throughout tongue. These results indicated that the tissue destruction caused by cryosurgery was large and suggest that the duration and frequency of freezing should be minimized for clinical use.

Gravity-driven microfluidic device placed on a slow-tilting table enables constant unidirectional perfusion culture of human induced pluripotent stem cells.

Gravity-driven microfluidics, which utilizes gravity force to drive liquid flow, offers portability and multi-condition setting flexibility because they do not require pumps or connection tubes to drive the flow. However, because the flow rate decreases with time in gravity-driven microfluidics, it is not suitable for stem cell experiments, which require long-term (at least a day) stability. In this study, gravity-driven microfluidics and a slow-tilting table were developed to culture cells under constant unidirectional perfusion. The microfluidic device was placed on a slow-tilting table, which tilts unidirectionally at a rate of approximately 7° per day to compensate for the reduction in the flow rate. Computational simulations showed that the pulsation of the flow arising from the stepwise movement of the table was less than 0.2%, and the flow was laminar. Hydrophilization of the tanks increased the flow rate, which is consistent with the theoretical values. We showed that vitronectin is better than laminin 511 fragments as a coating material for adhering human induced pluripotent stem cells on a microchamber made of polydimethylsiloxane, and succeeded in culturing the cells for 3 days. It is believed that the system offers easy-to-use cell culture tools, such as conventional multiwell culture vessels, and enables the control of the cell microenvironment.

Endodontic Infection-induced Inflammation Resembling Osteomyelitis of the Jaws in Toll-like Receptor 2/Interleukin 10 Double-knockout Mice.

INTRODUCTION: In general, mice develop chronic and nonhealing periapical lesions after endodontic infection. Surprisingly, we recently found that toll-like receptor 2 (TLR2)/interleukin 10 (IL-10) double-knockout (dKO) mice exhibited acute but resolving osteomyelitislike inflammation. In this study, we examined the kinetics of endodontic infection-induced inflammation in TLR2/IL-10 dKO mice and explored a potential mechanism of periapical wound healing mediated by the hypoxia-inducible factor 1 alpha (HIF-1α) subunit and arginase 1. METHODS: TLR2/IL-10 dKO and wild-type C57BL/6J mice were subjected to endodontic infection in the mandibular first molars. Mice were sacrificed on days 0 (noninfected), 10, and 21 postinfection. The extent of bone destruction, inflammation, bone deposition, and gene expression were determined by micro-computed tomographic imaging, histology, bone polychrome labeling, and microarray analysis. In addition, the effect of blocking endogenous HIF-1α was tested in infected TLR2/IL-10 dKO mice using the specific inhibitor YC-1. RESULTS: Infected TLR2/IL-10 dKO mice exhibited extensive bone destruction and inflammation on day 10 followed by spontaneous periapical wound healing including bone formation and resolution of inflammation by day 21 postinfection. In contrast, WT mice developed increasing chronic periapical inflammation over the 21-day observation period. Gene expression analyses and immunohistochemistry revealed that HIF-1α and arginase 1 were up-regulated in spontaneous wound healing in TLR2/IL-10 dKO mice. Blocking of HIF-1α in TLR2/IL-10 dKO mice using YC-1 resulted in significant inhibition of regenerative bone formation. CONCLUSIONS: The TLR2/IL-10 dKO mouse is a novel model resembling osteomyelitis of the jaws in which HIF-1α and arginase 1 appear to be crucial factors in spontaneous wound healing and bone repair.

Anatomical status of the human palatopharyngeal sphincter and its functional implications.

PURPOSE: The transition muscle between the palatopharyngeus (PP) and the superior constrictor of the pharynx (SCP) encircles the pharyngeal isthmus from behind and is designated as the palatopharyngeal sphincter (PPS). The PPS is inferred to play important roles for velopharyngeal closure, but its existence remains controversial and its roles have been regarded as being played by the SCP. The present study aimed to clarify the anatomical status and functional implications of the PPS. MATERIALS AND METHODS: Macroscopic and microscopic examinations were performed on 39 and 4 cadavers, respectively. In the former, the bilateral PPSs and their adjacent structures were exposed from outside and/or inside. In the latter, the velums embedded in paraffin were cut into frontal or sagittal sections and alternately processed with HE and Azan stains. RESULTS: The PPS originated from the nasal aspect of the lateral half of the palatine aponeurosis and the inferior margin of the medial pterygoid plate and was distinguishable from the PP descending in and along the palatopharyngeal arch and the cranialmost portion of the SCP in its origin. It passed dorsally on the lateral side of the levator veli palatini and traversed around the salpingopharyngeal fold running longitudinally. It then entered below the SCP and ran toward the pharyngeal raphe with SCP muscle fibers intermingled. CONCLUSIONS: The PPS is a muscle distinct from the SCP. Its contraction produces Passavant's ridge and conceivably enhances the efficiency of velopharyngeal closure by pressing the salpingopharyngeal fold and the musculus uvulae ridge against the velum.

Elevated CD14 (Cluster of Differentiation 14) and Toll-Like Receptor (TLR) 4 Signaling Deteriorate Periapical Inflammation in TLR2 Deficient Mice.

Apical periodontitis (periapical lesions) is an infection-induced chronic inflammation in the jaw, ultimately resulting in the destruction of apical periodontal tissue. Toll-like receptors (TLRs) are prominent in the initial recognition of pathogens. Our previous study showed that TLR4 signaling is proinflammatory in periapical lesions induced by a polymicrobial endodontic infection. In contrast, the functional role of TLR2 in regulation of periapical tissue destruction is still not fully understood. Using TLR2 deficient (KO), TLR2/TLR4 double deficient (dKO), and wild-type (WT) mice, we demonstrate that TLR2 KO mice are highly responsive to polymicrobial infection-induced periapical lesion caused by over activation of TLR4 signal transduction pathway that resulted in elevation of NF-kB (nuclear factor kappa B) and proinflammatory cytokine production. The altered TLR4 signaling is caused by TLR2 deficiency-dependent elevation of CD14 (cluster of differentiation 14), which is a co-receptor of TLR4. Indeed, neutralization of CD14 strikingly suppresses TLR2 deficiency-dependent inflammation and tissue destruction in vitro and in vivo. Our findings suggest that a network of TLR2, TLR4, and CD14 is a key factor in regulation of polymicrobial dentoalveolar infection and subsequent tissue destruction. Anat Rec, 299:1281-1292, 2016. © 2016 Wiley Periodicals, Inc.

Three-layered architecture of the popliteal fascia that acts as a kinetic retinaculum for the hamstring muscles.

When patients report pain in the popliteal fossa upon knee extension, the pain is usually localized in the lower region of the popliteal fossa. However, some patients complain of pain in the upper region of the popliteal fossa as the knee is flexed, which motivated us to examine the role of the popliteal fascia as the retinaculum of the hamstring muscles. Thirty-four thighs from 19 Japanese cadavers were dissected. The popliteal fascia was defined as the single aponeurotic sheet covering the popliteal fossa. We found that the fascia acted as a three-layered retinaculum for the flexor muscles of the thigh and provided a secure route for neurovascular structures to the lower leg in any kinetic position of the knee joint. The superficial layer of the popliteal fascia covering the thigh was strongly interwoven with the epimysium of biceps femoris along its lateral aspect and with that of the semimembranosus along its medial aspect, ensuring that the flexor muscles remained in their correct positions. The intermediate layer arose from the medial side of biceps femoris and merged medially with the superficial layer. The profound layer stretched transversely between the biceps femoris and the semimembranosus. Moreover, we investigated the nerve distribution in the popliteal fascia using Sihler's staining and whole-mount immunostaining for neurofilaments. The three-layered fascia was constantly innervated by branches from the posterior femoral cutaneous or saphenous nerve. The nerves were closely related and distributed to densely packed collagen fibers in the superficial layer as free or encapsulated nerve endings, suggesting that the fascia is involved in pain in the upper region of the popliteal fossa.

Establishment of immortalized mesenchymal stem cells derived from the submandibular glands of tdTomato transgenic mice.

Transgenic mice that overexpress the red fluorescent protein tdTomato (tdTomato mice) are well suited for use in regenerative medicine studies. Cultured cells from this murine model exhibit strong red fluorescence, enabling real-time in vivo imaging through the body surface of grafted animals. Mesenchymal stem cells (MSCs) have marked potential for use in cell therapy and regenerative medicine; however, the mechanisms that regulate their dynamics in vivo are poorly understood. In the present study, an MSC line was derived from the submandibular gland fibroblasts of tdTomato mice. The fluorescent signal from this cell line was observed in organs throughout the body, as well as in salivary glands. Primary culture cells derived from the submandibular gland were immortalized with SV40 large T antigen (GManSV cells); these cells exhibited increased migratory ability, as compared with those isolated from the sublingual gland. GManSV cells were tdTomato-positive and exhibited spindle-shaped fibroblastic morphology; they also robustly expressed mouse MSC markers: Stem cell antigen-1 (Sca-1), CD44, and CD90. This cell line retained multipotent stem cell characteristics, as evidenced by its ability to differentiate into both osteogenic and adipogenic lineages. These results indicate that Sca-1+/CD44+/CD90+-GManSV cells may be useful for kinetic studies of submandibular gland-derived MSCs in the context of in vitro co-culture with other types of salivary gland-derived cells. These cells may also be used for in vivo imaging studies, in order to identify novel cell therapy and regenerative medicine for the treatment of salivary gland diseases.

Anatomical status of the human musculus uvulae and its functional implications.

In our ongoing series of anatomical studies to determine the three-dimensional architecture of the human velar muscles, we have previously reported on the palatopharyngeus. The present study deals with the musculus uvulae (MU), in which the positional relationships of its origin to the posterior nasal spine and the palatine aponeurosis, as well as the interrelation between its anatomical status and functions, have yet to be clarified. Macroscopic and microscopic examinations were performed on 25 and 2 cadavers, respectively. In the former, bilateral MUs and their adjacent structures were exposed mainly from the nasal aspect. In the latter, the soft palates embedded in paraffin were cut into frontal and sagittal sections and alternately processed with HE and Azan stains. The left and right MUs adjacent to each other were found to run longitudinally along the midline beneath the nasal aspect of velum. It was overlaid by glandular tissue that increased in amount as it coursed distally. After originating from the oral surface of palatine aponeurosis, it ran backward to cross above the sling formed by the levator veli palatini muscles of both sides and reached the tip of uvula with its muscle fibers intermingled with glandular tissue. Past studies have proposed three functions of MU to enhance the efficiency of velopharyngeal closure: space occupier, stiffness modifier, and velar extensor. All of the above-described anatomical characteristics of MU could be explained as being adapted for these functions. This implies that MU is actively responsible for maintaining the velopharyngeal closure efficiency.

[Liver injury and hepatic encephalopathy induced by the herbal medicine Hochuekkito].

A 57-year-old man was admitted with pruritus and jaundice following treatment for fatigue with the herbal medicine Hochuekkito. The patient was prescribed prednisolone and ursodeoxycholic acid, but he developed progressive cholestasis that required intravenous methylprednisolone pulse therapy. After treatment with plasma exchange for prolonged prothrombin time, the patient recovered; however, his liver function deteriorated because of liver injury induced by trimethoprim-sulfamethoxazole for pneumocystis pneumonia. After reduction of trimethoprim-sulfamethoxazole, his liver function almost returned to normal by day 130 of admission. It has remained normal for 10 months since then. Therefore, when prescribing Hochuekkito, the possibility of drug-induced liver injury should be taken in account.

Improvement of pustulosis palmaris et plantaris by periodontal infection control in a patient with chronic periodontitis.

BACKGROUND: Pustulosis palmaris et plantaris (PPP) is a chronic, inflammatory, autoimmune-type disease characterized by sterile pustules of skin. The skin inflammation is influenced by several factors such as drugs, sunlight, metabolic and psychogenic factors as well as metal allergy. Here, we report a rare case that intensive periodontal treatment might have contributed to the improvement of skin inflammation. RESULTS: Skin inflammation regressed 1 month after intensive periodontal treatment. Both CD4/CD8 ratio and % of B cells in the blood sample were slightly decreased corresponding to the improvement of periodontal inflammation. CONCLUSIONS: Infection control of periodontal lesions might be one of attractive therapeutic targets in management of PPP.

Protective role of osteopontin in endodontic infection.

Endodontic infections are polymicrobial infections resulting in bone destruction and tooth loss. The host response to these infections is complex, including both innate and adaptive mechanisms. Osteopontin (OPN), a secreted, integrin-binding protein, functions in the regulation of immune responses and enhancement of leucocyte migration. We have assessed the role of OPN in the host response to endodontic infection using a well-characterized mouse model. Periapical bone loss associated with endodontic infection was significantly more severe in OPN-deficient mice compared with wild-type 3 weeks after infection, and was associated with increased areas of inflammation. Expression of cytokines associated with bone loss, interleukin-1alpha (IL-1alpha) and RANKL, was increased 3 days after infection. There was little effect of OPN deficiency on the adaptive immune response to these infections, as there was no effect of genotype on the ratio of bacteria-specific immunoglobulin G1 and G2a in the serum of infected mice. Furthermore, there was no difference in the expression of cytokines associated with T helper type 1/type2 balance: IL-12, IL-10 and interferon-gamma. In infected tissues, neutrophil infiltration into the lesion area was slightly increased in OPN-deficient animals 3 days after infection: this was confirmed by a significant increase in expression of neutrophil elastase in OPN-deficient samples at this time-point. We conclude that OPN has a protective effect on polymicrobial infection, at least partially because of alterations in phagocyte recruitment and/or persistence at the sites of infection, and that this molecule has a potential therapeutic role in polymicrobial infections.

Long root of deciduous anterior teeth.

A 24-year-old female Japanese patient presented with remarkably long roots of retained deciduous anterior teeth and permanent anterior teeth in the upper and lower jaw. Four lower anterior teeth were extracted for esthetic reasons. The patient had no apparent clinical syndrome related to the teeth or jaw, nor did there appear to be a family history of this condition. The extracted teeth and their lengths were as follows: the lower right deciduous lateral incisor was 25.55 mm long (root length, 18.95 mm); the lower left deciduous lateral incisor was 22.10 mm long (root length, 17.25 mm); the lower right deciduous canine was 27.95 mm long (root length, 20.60 mm); and the lower left deciduous canine was 23.90 mm long (root length, 17.65 mm).

Th1 biased response to a novel Porphyromonas gingivalis protein aggravates bone resorption caused by this oral pathogen.

In previous studies we showed that biasing the immune response to Porphyromonas gingivalis antigens to the Th1 phenotype increases inflammatory bone resorption caused by this organism. Using a T cell screening strategy we identified eight P. gingivalis genes coding for proteins that appear to be involved in T-helper cell responses. In the present study, we characterized the protein encoded by the PG_1841 gene and evaluated its relevance in the bone resorption caused by P. gingivalis because subcutaneous infection of mice with this organism resulted in the induction of Th1 biased response to the recombinant PG1841 antigen molecule. Using an immunization regime that strongly biases toward the Th1 phenotype followed by challenge with P. gingivalis in dental pulp tissue, we demonstrate that mice pre-immunized with rPG1841 developed severe bone loss compared with control immunized mice. Pre-immunization of mice with the antigen using a Th2 biasing regime resulted in no exacerbation of the disease. These results support the notion that selected antigens of P. gingivalis are involved in a biased Th1 host response that leads to the severe bone loss caused by this oral pathogen.

Mast cell-specific gangliosides and FcepsilonRI follow the same endocytic pathway from lipid rafts in RBL-2H3 cells.

Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in FcepsilonRI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with FcepsilonRI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against FcepsilonRI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as FcepsilonRI and at 3 hr are present in the dense lysosome fraction. Using transmission electron microscopy, with antibody against the gangliosides conjugated to horseradish peroxidase and antibody against FcepsilonRI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and FcepsilonRI are internalized in the same coated vesicles. At 5 min, the gangliosides and FcepsilonRI can be identified in early endosomes and at 3 hr are found together in acid phosphatase-positive lysosomes. This study demonstrates that the mast cell-specific gangliosides are internalized from lipid rafts in the same vesicles and traffic intracellularly with the same kinetics as FcepsilonRI. This study contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Rare case of the inferior mesenteric artery and the common hepatic artery arising from the superior mesenteric artery.

We found a case in which inferior mesenteric artery and the common hepatic artery arose from the superior mesenteric artery, forming the common hepatomesenteric trunk, during a routine dissection carried out at Iwate Medical University in 2002. This variation is rare, but can be embryonically explained. A change in the positions of the disappearance of the ventral splanchnic arteries and the longitudinal anastomotic channel results in variations in the system of arteries distributed to the digestive organs. In the present case, the longitudinal anastomotic channel between the superior and the inferior mesenteric arteries survived to form the common mesenteric artery, which was joined by the common hepatic artery, forming the common hepatomesenteric trunk.

Abnormal anterior belly of the digastric muscle: a proposal for the classification of abnormalities.

We recognized an abnormal anterior belly of the digastric muscle in an 83-year-old male cadaver. Three muscle bundles were observed on the left anterior belly: (i) attached to the left digastric fossa; (ii) attached to the right digastric fossa; and (ii) attached to the raphe of the mylohyoid muscle. Four muscle bundles were recognized on the right anterior belly: (i) attached to raphe of the mylohyoid muscle; (ii, iii) attached to the exterior surface on the base of the mandible from the raphe of the mylohyoid muscle; and (iv) attached to the interior surface on the base of the mandible from the raphe of the mylohyoid muscle. The raphe of the mylohyoid muscle was curved significantly to right and the four abnormal bundles found on the right anterior belly (see above) were attached to its curved point.

Analysis of the three-dimensional lymphatic architecture of the periodontal tissue using a new 3D reconstruction method.

Few studies of lymphatic vessels have been reported because diacrisis of this vascular system is rare. A complete examination of diacrisis of venula is not yet possible even using recent enzyme-histochemical or immunohistochemical techniques. In this study, we examined a lymphatic vessel by serial sectioning from the afferent lymphatic of the lymph node to the periphery by three-dimensional observation using a three-dimensional reconstitution method. This method was conventional and accurate, and the time required for processing was markedly reduced by using a computer. Reconstitution of the vasculature became possible utilizing the entire section instead of a portion of a parenchymatous organ. We examined the lymphatic vessel architecture of various oral regions, including gingiva, tongue, and the floor of the mouth, using this method. In the future, using this method, we plan to investigate the alteration of lymphatic vessel architecture in a pathological region, and correlate these changes with the dynamics of lymphatic vessel absorption.