Oligodendrocyte precursor cells support blood-brain barrier integrity via TGF-ß signaling.

Trophic coupling between cerebral endothelium and their neighboring cells is required for the development and maintenance of blood-brain barrier (BBB) function. Here we report that oligodendrocyte precursor cells (OPCs) secrete soluble factor TGF-ß1 to support BBB integrity. Firstly, we prepared conditioned media from OPC cultures and added them to cerebral endothelial cultures. Our pharmacological experiments showed that OPC-conditioned media increased expressions of tight-junction proteins and decreased in vitro BBB permeability by activating TGB-ß-receptor-MEK/ERK signaling pathway. Secondly, our immuno-electron microscopic observation revealed that in neonatal mouse brains, OPCs attach to cerebral endothelial cells via basal lamina. And finally, we developed a novel transgenic mouse line that TGF-ß1 is knocked down specifically in OPCs. Neonates of these OPC-specific TGF-ß1 deficient mice (OPC-specific TGF-ß1 partial KO mice: PdgfraCre/Tgfb1flox/wt mice or OPC-specific TGF-ß1 total KO mice: PdgfraCre/Tgfb1flox/flox mice) exhibited cerebral hemorrhage and loss of BBB function. Taken together, our current study demonstrates that OPCs increase BBB tightness by upregulating tight junction proteins via TGF-ß signaling. Although astrocytes and pericytes are well-known regulators of BBB maturation and maintenance, these findings indicate that OPCs also play a pivotal role in promoting BBB integrity.

Analysis of MRI findings in minimum invasive treatment for habitual temporomandibular joint dislocation by autologous blood injection around the temporomandibular joint capsule.

The aim of this study was to investigate magnetic resonance imaging (MRI) findings following autologous blood injection (ABI) for habitual temporomandibular joint (TMJ) dislocation. MRI was performed one hour and four and twelve weeks after ABI, revealing three types of significant findings. The first type was similar to hematoma and/or joint effusion in the articular capsule of the TMJ (type I). The second showed sporadic and diffuse T2 emphasis around the TMJ capsule (type II). The third involved a decreased range of condyle movement compared to before ABI (type III). Furthermore, we analyzed the three types of significant MRI findings. At one hour after ABI, type I was Grade 0 in 0 of 14 patients, Grade 1 in 8, Grade 2 in 2, and Grade 3 in 4. Type II was seen in 9 of the 14 cases and type III in 8. After twelve weeks, all cases of type I were Grade 0, no type II cases were evident, and type III was seen in 11 cases. Injecting autologous blood into surrounding TMJ tissues is an important factor in ABI. Minimally invasive treatment for habitual TMJ dislocation using ABI around the TMJ capsule appears to represent a very effective and safe treatment.

Effects of platform switching on crestal bone around implants: a histomorphometric study in monkeys.

A number of human comparison studies and case series have demonstrated the effectiveness of using a platform-switched implant-to-abutment connection to prevent peri-implant bone loss and subsequent soft tissue loss. To compare the bone around platform-switched and nonplatform-switched connections, adjacent pairs of both connection types were placed in a one-stage surgical procedure on each side of the mandibles of three monkeys (Macaca fascicularis). Eight weeks after abutment connection, histomorphometric analysis showed that both vertical and horizontal alveolar bone resorption had occurred around the nonplatform-switched implants, whereas bone was maintained vertically and new bone formed horizontally around the platform-switched implants.

Acrylic resin injection method for blood vessel investigations.

The injection of acrylic resin into vessels is an excellent method for macroscopically and microscopically observing their three-dimensional features. Conventional methods can be enhanced by removal of the polymerization inhibitor (hydroquinone) without requiring distillation, a consistent viscosity of polymerized resin, and a constant injection pressure and speed. As microvascular corrosion cast specimens are influenced by viscosity, pressure, and speed changes, injection into different specimens yields varying results. We devised a method to reduce those problems. Sodium hydroxide was used to remove hydroquinone from commercial methylmethacrylate. The solid polymer and the liquid monomer were mixed using a 1 : 9 ratio (low-viscosity acrylic resin, 9.07 ± 0.52 mPa•s) or a 3:7 ratio (high-viscosity resin, 1036.33 ± 144.02 mPa•s). To polymerize the acrylic resin for injection, a polymerization promoter (1.0% benzoyl peroxide) was mixed with a polymerization initiator (0.5%, N, N-dimethylaniline). The acrylic resins were injected using a precise syringe pump, with a 5-mL/min injection speed and 11.17 ± 1.60 mPa injection pressure (low-viscosity resin) and a 1-mL/min injection speed and 58.50 ± 5.75 mPa injection pressure (high-viscosity resin). Using the aforementioned conditions, scanning electron microscopy indicated that sufficient resin could be injected into the capillaries of the microvascular corrosion cast specimens.

In vitro construction of scaffold-free bilayered tissue-engineered skin containing capillary networks.

Many types of skin substitutes have been constructed using exogenous materials. Angiogenesis is an important factor for tissue-engineered skin constructs. In this study, we constructed a scaffold-free bilayered tissue-engineered skin containing a capillary network. First, we cocultured dermal fibroblasts with dermal microvascular endothelial cells at a ratio of 2 : 1. A fibrous sheet was formed by the interactions between the fibroblasts and the endothelial cells, and capillary-like structures were observed after 20 days of coculture. Epithelial cells were then seeded on the fibrous sheet to assemble the bilayered tissue. HE staining showed that tissue-engineered skin exhibited a stratified epidermis after 7 days. Immunostaining showed that the epithelium promoted the formation of capillary-like structures. Transmission electron microscopy (TEM) analysis showed that the capillary-like structures were typical microblood vessels. ELISA demonstrated that vascularization was promoted by significant upregulation of vascularization associated growth factors due to interactions among the 3 types of cells in the bilayer, as compared to cocultures of fibroblast and endothelial cells and monocultures.

Classification of persistent left superior vena cava considering presence and development of both superior venae cavae, the anastomotic ramus between superior venae cavae, and the azygos venous system.

Subjects with persistent left superior vena cava were classified on the basis of the presence and thickness of both superior venae cavae, the anastomotic ramus between the superior venae cavae (anastomotic ramus), and the presence of both azygos veins. Among subjects with persistent left superior vena cava, the percentage of those with weak development of the anastomotic ramus (41.5 %) or absence of an anastomotic ramus (35.8 %) was 77.3 %. In addition, 54.7 % of subjects had a left azygos vein. However, 88.7 % of subjects had a right azygos vein. In this classification, the most frequently observed types included the presence of both superior venae cavae, an anastomotic ramus, and both azygos veins (20.8 %). During student dissection practice sessions performed on 337 cadavers that were carried out from 2002 through 2010, a subject having a left superior vena cava (in 2002) and a subject having both superior venae cavae (in 2003) were detected. The former case was reported previously. The latter case is reported in this paper. The incidence of persistent left superior vena cava was 0.59 % (2/337 cadavers).

Morphological study of the palatal gingiva of the maxillary first molar in the type 2 diabetes mellitus model rat.

SUMMARY: We studied morphological changes at the maxillary first molar in a model rat for type 2 spontaneous diabetes mellitus (DM), the Goto-Kazizaki (GK) rat, vs. the normal 8-week-old Wistar rat. Serial frontal sections of the gingiva of the maxilla with the bone were prepared from the rats. Image analyses, performed on light micrographs of the hematoxylin-eosin stained specimens, allowed comparison of the thickness of the keratinized, granular, prickle, and basal layers. In addition, the cell population of the granular and prickle layers and the cross-sectional area of the connective tissue beneath the mucosal epithelium were examined. The thickness of the capillary of the maxillary first molar was determined by image analysis of scanning electron micrographs of microvascular corrosion cast specimens. We found that the thickness of the keratinized, granular, and prickle layers was significantly higher in the DM vs. normal group, as were the cell population of the granular and prickle layers. In contrast, the cross-sectional area of the connective tissue beneath the mucosal epithelium, and the thickness of the capillary were significantly lower in the DM vs. normal sections. Therefore, we consider that the DM-associated hyperglycemia causes hypertrophy of the mucosal epithelium, atrophy of the connective tissue beneath the mucosal epithelium, and microangiopathy of the capillary of the palatal gingiva of the maxillary first molar in the GK rat.

Comparative study of the ramification patterns of the subclavian branches as the subclavian artery passes in front or behind the scalenus anterior muscle.

We investigated the ramification patterns of four subclavian branches (i.e., vertebral artery, internal thoracic artery, thyrocervical trunk, and costocervical trunk) as the subclavian artery passes in front or behind the scalenus anterior muscle. The investigation was carried out on 56 cadavers (112 cases) during student dissection practice sessions at Osaka Dental University. In 110 of the 112 cases, the subclavian artery passed behind the scalenus anterior muscle. The pattern of ramification of the subclavian branches in these cases was classified into six types (types A-F). In the remaining two cases (two cadavers), the subclavian artery passed in front of the scalenus anterior muscle. In both of these latter cases, the pattern of ramification of the subclavian branches differed from the six pattern types observed as the subclavian artery passed behind the scalenus anterior muscle: the first branch was the vertebral artery; the second, the costocervical trunk; the third, the thyrocervical trunk; the fourth, the internal thoracic artery. This same pattern of ramification was observed in three previously reported cases (two cadavers) in which the subclavian artery passed in front of the scalenus anterior muscle. Taken together, these observations indicate that the ramification pattern reported here and in a previous investigation for the subclavian artery passing in front of the scalenus anterior muscle is characteristic of this anatomical condition.

Left superior vena cava with left azygos vein.

An incidence of a left superior vena cava with a left azygos vein was found in a cadaver during an Osaka Dental University student's dissection practice session in 2002. The left superior vena cava began from the confluence between the right and left brachiocephalic veins, and extended directly into the left superior edge of the coronary sinus. No right superior vena cava was observed. The left azygos vein, receiving the bilateral intercostal veins, extended into the left superior vena cava. The developmental mechanism of the left superior vena cava was considered as follows; because the proximal part of the left anterior cardinal vein and the left common cardinal vein remained, and the proximal part of the right anterior cardinal vein and the right common cardinal vein degenerated and disappeared. The developmental mechanism of the left azygos vein was considered as follows; because the proximal part of the left posterior cardinal vein remained, and the root of the azygos vein was formed.

Cementogenesis examined from the viewpoint of microcirculation.

The purpose of this study was to perform a three-dimensional observation, via microvascular corrosion casts, of the microcirculation system during deposition of cementum after flap surgery and to investigate the permeable structure of the vascular endothelium. Two stages of wound healing after flap surgery were confirmed based on successive vascular changes. The transition between these stages occurred 3 weeks after surgery, at which time new blood vessels disappeared and an early stage of accumulation of new cementum was apparent. Hence, fibrous repair occurred during the first stage, and repair of hard tissue (ie, formation of cementum) occurred during the second stage. These findings suggest that metabolic activity in cementogenesis is low, based on the condition of the blood vessels, and therefore new cementum is not easily formed.

Morphological study of the lingual papillae in the ferret (Mustela putorius furo).

We used four ferrets (Mustela putorius furo) and observed these animals dorsal tongue surface morphology via scanning electron microscope and light microscope. In this investigation, we focused on the food habits and discussed the morphology of the lingual papillae from the viewpoint of comparative anatomy. The ferret has conically-shaped filiform papillae in the posterior, middle and anterior region of the tongue body, and circular-distributed filiform papillae in the lingual apex region. The ferret has fungiform papillae with hemispheric shaped summits in the posterior and middle region with square-shaped summits in the anterior and the lingual apex region. The ferret has V-shaped vallate papillae with eight papillae in two lines or 12 papillae in three lines on the tongue root. No foliate papillae were observed on the dorsal tongue surface of the ferret. The ferret belongs to the carnivore family but has a highly developed vallate papillae which are taste bud papillae and many taste glands. Thus we conclude that the ferrets need a large amount of saliva to swallow food because it demonstrates a large number of taste glands.

Morphological study of the connective tissue papillae and the capillary loops on the lingual dorsum in the type 2 diabetes mellitus model rats.

We performed a morphological study in type 2 diabetes mellitus (DM) model rats by focusing on the connective tissue papillae (CTPe) and capillary loops (CLs) of filiform papillae in the lingual dorsum in the type 2 DM model rats (GK rats). The normal group comprised nine 8-week-old male Wistar rats. The DM group comprised nine 8-week-old male GK rats. Image analysis of light microscopic specimens in CTPe was performed (height and cross-sectional area). Image analysis of scanning electron microscopic specimens (acrylic plastic injection method) in CLs was performed (number, thickness, height, interval between the tops of the two CLs, and crossing ratio between ascending and descending crura (CRC)). We compared these values between both groups (Student's t test). The former analysis revealed that the height and cross-sectional area of CTPe were smaller in the DM group than in the normal group. The latter analysis revealed that the thickness, height, and CRC of the CLs were smaller in the DM group than in the normal group. However, no significant differences were detected in the number and interval of CLs between both groups. Therefore, we concluded that DM caused regressive change in CTPe and CLs.

Influence of age-related changes in nitric oxide synthase-expressing neurons in the rat supraoptic nucleus on inhibition of salivary secretion.

Age-related inhibition of salivary secretion has been demonstrated in rats, and the nitric oxide (NO) present in the supraoptic nucleus (SON) and the medial septal area has been reported to play an inhibitory role in the regulation of salivary secretion. In the present study, we investigated the age-related changes occurring in the NO synthase (NOS)-expressing neurons in the SON, which is related to the production of NO, and discussed the interrelation between the age-related changes in the NOS-expressing neurons and the age-related inhibition of salivary secretion. Nissl staining and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were performed for young adult and aged rats. Quantitative analysis was also performed using the Nissl-stained and NADPH-d-positive neurons. Although the numbers of the Nissl-stained neurons did not change, significant age-related increases were detected in cell number, cell size and reactive density of the NADPH-d-positive neurons. Therefore, the production of NO in the SON neurons increased with age. We concluded that the age-related increase in the NO in the SON might be a factor that contributes to the age-related inhibition of salivary secretion.

Bilateral subclavian arteries passing in front of the scalenus anterior muscles.

Herein, we present a very rare case of bilateral subclavian arteries passing in front of the scalenus anterior muscles in a cadaver. This abnormality was observed in a 73-year-old Japanese male cadaver during a dissection session for students in 2004 at Osaka Dental University. The bilateral scalenus anterior muscle originated from the anterior tubercle of the transverse processes of the fifth and sixth cervical vertebrae and inserted into the scalene tubercle of the first ribs. The right scalenus minimus muscle was observed, but no left scalenus minimus muscle was observed. The aortic arch was a type A according to Adachi's classification. The origin of the internal thoracic artery was distal to that of the thyrocervical trunk. The bilateral brachial plexuses was formed by the union of the ventral rami from the fifth cervical to the first thoracic nerves and passed between the scalenus anterior and the scalenus medius muscles. To our knowledge, such a case has not been reported previously.

Reconstruction of a tissue-engineered skin containing melanocytes.

The objective of this study was to establish a new method for reconstruction of a tissue-engineered skin containing melanocytes by employing tissue engineering. The keratinocytes, melanocytes and dermal fibroblasts were isolated and purified from human foreskin biopsies. Then the cells were used to construct a tissue-engineered skin containing melanocytes. The localization of melanocytes in the tissue-engineered skin was detected by DOPA staining, S-100 immunohistochemical staining and transmission electron microscope (TEM). The results showed that the melanocytes could be detected in the basal layer of the constructed skin and the melanocytes showed dendritic morphology. Moreover the constructed skins were used to repair the athymic mice skin defects. Animal experiment results indicated that the skin equivalents could successfully repair full thickness skin defects in athymic mice and generated black skins by 6weeks after grafting. Melanocytes located in the basal layer of the athymic mice skin could also be detected by using the S-100 immunohistochemical staining. Our established method is useful to repair the full-thickness skin defects.

The role of bone morphogenetic protein-2 in vivo in regeneration of peripheral nerves.

We investigated the effects of bone morphogenetic protein-2 (BMP-2) and some other BMPs on regeneration of peripheral motor nerves in vivo. The facial nerves of 24 New Zealand rabbits were crushed to examine a series of retrograde changes in the facial nuclei and axons, in what has been called the "axon reaction". The facial nerves of the experimental group were treated with epineurial coaptation and BMP-2 after the injury. Nerves not treated with BMP-2 were regarded as controls. The expression of BMP-2 was investigated by in situ hybridisation in the neurons of facial nuclei. The electrophysiology, image analysis and transmission electron microscopy were used to evaluate the level of the recovery of facial nerves. The results showed that the axons in the experimental group were thicker and denser than those in the control group four weeks later. The expression of BMP-2 in the neurons of facial nuclei increased after injury. The electron microscopic observations showed that the axons' degeneration in the experimental group was less than that in the control group. Despite the morphological difference between the two groups, there was no apparent difference between them in nerve conduction velocity. These findings suggest that BMP-2 might be involved in the regeneration of facial nerves, and might function as a potential neurotrophic factor.

Morphological interrelationship between astrocytes and nerve endings in the rat spinal trigeminal nucleus caudalis.

It has been reported that the spinal trigeminal nucleus caudalis (Sp5C), which receives nociceptive information from the oro-facial regions, has four laminae. To clarify the role of glial cells in the transmission of the nociceptive information, the present study was conducted to examine the detailed distribution of astrocytes in each lamina and also to investigate a morphological interrelationship between the astrocytes and nerve endings in the rat Sp5C. After the preparation of the serial cryostat sections, immunohistochemistry for glial fibrillary acidic protein (GFAP) was employed to identify the astrocytes, and immunohistochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), was used for the nerve endings. We also employed double-labeling immunofluorescence and electron microscopic immunohistochemistry for the GFAP/SP or GFAP/CGRP. GFAP-positive reactions were observed in all laminae of the Sp5C, and SP- or CGRP-positive nerve endings were observed in the lamina I and II. Additionally, we clarified the presence of GFAP/SP- or GFAP/CGRP-positive reactions by the double-labeling immunofluorescence and demonstrated the morphological interrelationship between the astrocytes and nerve endings by the double-labeling electron microscopic immunohistochemistry. These findings suggest that astrocytes might play some roles in the transmission of nociceptive information from the oro-facial region.

A case of right partial and double internal jugular veins.

We present a very rare case of right partial and double internal jugular veins, found in an 86-year-old Japanese female cadaver during a student dissection practice session in 2002 at Osaka Dental University. In this case, the right internal jugular vein separated into medial and lateral branches at a level with the middle of the fifth cervical vertebra. Both branches had the same thickness as an internal jugular vein and poured into the right subclavian vein. A slender venous space slit was formed by these two branches and the right subclavian vein. The inferior belly of the right omohyoid muscle and the inferior root of the right ansa cervicalis passed through the superior region in this venous space. To our knowledge, this case has never been reported previously. Therefore, we attempted to investigate the incidence based on existing references for similar cases and speculated on the development based on our findings. We considered the medial branch was the right internal jugular vein and the lateral branch was the communicating branch between the external and internal jugular veins.

Age-related changes in oxytocin-, arginine vasopressin- and nitric oxide synthase-expressing neurons in the supraoptic nucleus of the rat.

Many histochemical investigations indicated that the oxytocin (OXY), the arginine vasopressin (AVP) and the nitric oxide synthase (NOS) have been synthesized in the supraoptic nucleus (SON) neurons. The objective of this study was to examine the age-related expression of the OXY, the AVP and the NOS in the SON of the young adult (2-month-old) and the aged (24-month-old) rats. The histochemistry for reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d; marker for the NOS) and the double labeling histochemistry for the OXY/NADPH-d or the AVP/NADPH-d were employed, and the quantitative analysis was performed with a computer-assisted image processing system. In comparison of the young adult and the aged group, the cell number, the cell size and the reactive density of the NOS-expressing neurons showed a significant increase along with age, and these evidences suggested the age-related increase of the nitric oxide (NO) production. The age-related significant increase was not detected in the number of the OXY/NOS-expressing neurons in the dorsal part, but was detected in the number of the AVP/NOS-expressing neurons in the ventral part. Based on our histochemical findings and reports demonstrated by other authors, we attempted to discuss the physiological role of NOS for the secretion of posterior pituitary hormones along with age.

Microvascular response in the periosteum following mucoperiosteal flap surgery in dogs: 3-dimensional observation of an angiogenic process.

BACKGROUND: An increase in blood flow from the periosteum after mucoperiosteal flap surgery is essential for healing and angiogenesis and repair may work in close cooperation to facilitate this process. To investigate the role of the periosteal vascular plexus in the healing process, we used 3-dimensional (3-D) and ultrastructural monitoring of the angiogenic process after elevation of the mucoperiosteal flap. METHODS: Mucoperiosteal flap surgery was performed on nine adult beagle dogs. The periosteal vascular plexus was observed 3, 5, and 7 days after surgery in histological specimens in which blood vessels were injected with India ink under a light microscope, in ultrathin sections under a transmission electron microscope, and in acryl plastic vascular cast specimens under a scanning electron microscope. RESULTS: On day 3 after surgery, new blood vessels, formed through sprouting, bridging, and intussusception, were observed in ultrathin sections and vascular casts. In addition, blood island-like structures consisting of clustered immature endothelial cells were noted in the repaired tissue. On days 5 to 7 after surgery, 3-D observation of vascular casts clarified that these new blood vessels had a sinus-like morphology in the interstitium of the periosteal vascular plexus. These new sinusoidal vessels exhibited a stereoscopic structure with increased continuity as the blood vessels matured and ultrastructurally the vascular endothelium was thinned. CONCLUSIONS: After mucoperiosteal flap elevation, the periosteal vasculature exhibited potent blood vessel-forming activity through various angiogenic mechanisms and through repair activity. Our results provide a 3-dimensional clarification that the periosteal vascular plexus has an important role in the healing process after flap surgery.