Yoshihito Shimazu

Identification of mesenchymal stem cell (MSC)‐transcription factors by microarray and knockdown analyses, and signature molecule‐marked MSC in bone marrow by immunohistochemistry

Although ex vivo expanded mesenchymal stem cells (MSC) have been used in numerous studies, the molecular signature and in vivo distribution status of MSC remain unknown. To address this matter, we identified numerous human MSC‐characteristic genes—including nine transcription factor genes —using DNA microarray and real‐time RT‐PCR analyses: Most of the MSC‐characteristic genes were down‐regulated 24 h after incubation with osteogenesis‐, chondrogenesis‐ or adipogenesis‐induction medium, or 48–72 h after knockdown of the nine transcription factors. Furthermore, knockdowns of ETV1, ETV5, FOXP1, GATA6, HMGA2, SIM2 or SOX11 suppressed the self‐renewal capacity of MSC, whereas those of FOXP1, SOX11, ETV1, SIM2 or PRDM16 reduced the osteogenic‐ and/or adipogenic potential. In addition, immunohistochemistry using antibodies for the MSC characteristic molecules—including GATA6, TRPC4, FLG and TGM2—revealed that MSC‐like cells were present near the endosteum and in the interior of bone marrow of adult mice. These findings indicate that MSC synthesize a set of MSC markers in vitro and in vivo, and that MSC‐characteristic transcription factors are involved in MSC stemness regulation.

Enamel Mineralization and an Initial Crystalline Phase

In this communication, we summarized our recent experimental approaches to an unsettled issue, i.e., the nature and role of an acidic precursor in enamel mineralization. The objectives we specially focused our attention on are: the composition, structure and high resolution images of enamel crystals at various developmental stages, thermodynamic and kinetic consideration of octacalcium phosphate (OCP) vs hydroxyapatite (HA) precipitation in physiological media simulating the enamel fluid, reversible changes in the composition and structure of OCP, effects of fluoride at low concentrations and enamel proteins on OCP hydrolysis, and adsorption of enamel proteins onto OCP and fluoridated hydrolysates at neutral pH and room temperature. On the basis of all experimental evidence, we propose that enamel crystal growth comprises two events: the two-dimensional growth of an OCP-like precursor in a narrow outermost zone adjacent to the ameloblasts and the subsequent overgrowth of apatite units on the template under discrete fluid environment in the underlying region distant from the cell layer. The experimental data also support the concept that the whole process of enamel mineralization is modulated substantially through interaction between enamel proteins and crystals including the acidic precursor.

Systemic administration of resveratrol suppress the nociceptive neuronal activity of spinal trigeminal nucleus caudalis in rats

Although a modulatory role has been reported for the red wine polyphenol resveratrol on several types of ion channels and excitatory synaptic transmission in the nervous system, the acute effects of resveratrol in vivo, particularly on nociceptive transmission of the trigeminal system, remain to be determined. The aim of the present study was to investigate whether acute intravenous resveratrol administration to rats attenuates the excitability of wide dynamic range (WDR) spinal trigeminal nucleus caudalis (SpVc) neurons in response to nociceptive and non-nociceptive mechanical stimulation in vivo. Extracellular single unit recordings were made from 18 SpVc neurons in response to orofacial mechanical stimulation of pentobarbital-anesthetized rats. Responses to both non-noxious and noxious mechanical stimuli were analyzed in the present study. The mean firing frequency of SpVc WDR neurons in response to both non-noxious and noxious mechanical stimuli was inhibited by resveratrol (0.5-2 mg/kg, i.v.) and maximum inhibition of the discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 10 min. These inhibitory effects were reversed after approximately 20 min. The relative magnitude of inhibition by resveratrol of SpVc WDR neuronal discharge frequency was significantly greater for noxious than non-noxious stimulation. These results suggest that, in the absence of inflammatory or neuropathic pain, acute intravenous resveratrol administration suppresses trigeminal sensory transmission, including nociception, and so resveratrol may be used as a complementary and alternative medicine therapeutic agent for the treatment of trigeminal nociceptive pain, including hyperalgesia.

Resveratrol attenuates inflammation-induced hyperexcitability of trigeminal spinal nucleus caudalis neurons associated with hyperalgesia in rats

Background Resveratrol, a component of red wine, has been reported to decrease prostaglandin E2 production by inhibiting the cyclooxygenase-2 cascade and to modulate various voltage-dependent ion channels, suggesting that resveratrol could attenuate inflammatory hyperalgesia. However, the effects of resveratrol on inflammation-induced hyperexcitability of nociceptive neurons in vivo remain to be determined. Thus, the aim of the present study was to determine whether daily systemic administration of resveratrol to rats attenuates the inflammation-induced hyperexcitability of spinal trigeminal nucleus caudalis wide-dynamic range neurons associated with hyperalgesia. Results Inflammation was induced by injection of complete Freund’s adjuvant into the whisker pad. The threshold of escape from mechanical stimulation applied to whisker pad in inflamed rats was significantly lower than in control rats. The decreased mechanical threshold in inflamed rats was restored to control levels by daily systemic administration of resveratrol (2 mg/kg, i.p.). The mean discharge frequency of spinal trigeminal nucleus caudalis wide-dynamic range neurons to both nonnoxious and noxious mechanical stimuli in inflamed rats was significantly decreased after resveratrol administration. In addition, the increased mean spontaneous discharge of spinal trigeminal nucleus caudalis wide-dynamic range neurons in inflamed rats was significantly decreased after resveratrol administration. Similarly, resveratrol significantly diminished noxious pinch-evoked mean after discharge frequency and occurrence in inflamed rats. Finally, resveratrol restored the expanded mean size of the receptive field in inflamed rats to control levels. Conclusion These results suggest that chronic administration of resveratrol attenuates inflammation-induced mechanical inflammatory hyperalgesia and that this effect is due primarily to the suppression of spinal trigeminal nucleus caudalis wide dynamic range neuron hyperexcitability via inhibition of both peripheral and central cyclooxygenase-2 cascade signaling pathways. These findings support the idea of resveratrol as a potential complementary and alternative medicine for the treatment of trigeminal inflammatory hyperalgesia without side effects.

Local administration of resveratrol inhibits excitability of nociceptive wide-dynamic range neurons in rat trigeminal spinal nucleus caudalis

Although we recently reported that intravenous administration of resveratrol suppresses trigeminal nociception, the precise peripheral effect of resveratrol on nociceptive and non-nociceptive mechanical stimulation-induced trigeminal neuron activity in vivo remains to be determined. The aim of the present study was to investigate whether local subcutaneous administration of resveratrol attenuates mechanical stimulation-induced excitability of trigeminal spinal nucleus caudalis (SpVc) neuron activity in rats, in vivo. Extracellular single-unit recordings were made of SpVc wide-dynamic range (WDR) neuron activity in response to orofacial mechanical stimulation in pentobarbital-anesthetized rats. Neurons responded to non-noxious and noxious mechanical stimulation applied to the orofacial skin. Local subcutaneous administration of resveratrol (1-10mM) into the orofacial skin dose dependently and significantly reduced the mean number of SpVc WDR neurons firing in response to both non-noxious and noxious mechanical stimuli, with the maximal inhibition of discharge frequency in response to both stimuli being seen within 5min. These inhibitory effects were no longer evident after approximately 20min. The mean magnitude of inhibition by resveratrol (10mM) of SpVc neuron discharge frequency was almost equal to that of the local anesthetic 1% lidocaine (37mM). These results suggest that local injection of resveratrol into the peripheral receptive field suppresses the excitability of SpVc neurons, possibly via inhibition of Na(+) channels in the nociceptive nerve terminals of trigeminal ganglion neurons. Therefore, local subcutaneous administration of resveratrol may provide relief of trigeminal nociceptive pain, without side effects, thus contributing to the suite of complementary and alternative medicines used as local anesthetic agents.

Three-Dimensional Reconstruction of Oral Tongue Squamous Cell Carcinoma at Invasion Front

We conducted three-dimensional (3D) reconstruction of oral tongue squamous cell carcinoma (OTSCC) using serial histological sections to visualize the architecture of invasive tumors. Fourteen OTSCC cases were collected from archival paraffin-embedded specimens. Based on a pathodiagnostic survey of whole cancer lesions, a core tissue specimen (3 mm in diameter) was dissected out from the deep invasion front using a paraffin tissue microarray. Serial sections (4 μm thick) were double immunostained with pan-cytokeratin and Ki67 antibodies and digitized images were acquired using virtual microscopy. For 3D reconstruction, image registration and RGB color segmentation were automated using ImageJ software to avoid operator-dependent subjective errors. Based on the 3D tumor architecture, we classified the mode of invasion into four types: pushing and bulky architecture; trabecular architecture; diffuse spreading; and special forms. Direct visualization and quantitative assessment of the parenchymal-stromal border provide a new dimension in our understanding of OTSCC architecture. These 3D morphometric analyses also ascertained that cell invasion (individually and collectively) occurs at the deep invasive front of the OTSCC. These results demonstrate the advantages of histology-based 3D reconstruction for evaluating tumor architecture and its potential for a wide range of applications.

Hertwig's Epithelial Cells and Multi-root Development of Molars in Mice

Abstract Previously, we focused on multi-root development of the upper and lower molars in mice with the aid of three-dimensional (3D) reconstruction technologies. ICR mice, 3 to 28 days old, were used. The upper and lower jaws including the molars were first used for the collection of micro-computed tomography (μCT) data and, consecutively, were processed to prepare paraffin-embedded sagittal serial sections. Timesequential morphogenesis of the upper and lower first molars was monitored using, μCT-3D models, edited in motion view by superimposing 3D models made at different ages. Histology-based 3D reconstruction in conjunction with immunohistochemistry using cytokeratin as a marker of epithelial cells was also emloyed to visualize the Hertwigs epithelial root sheath (HERS)-guided closure of the pulp chamber floor and the latter-stage disintegration and fate of HERS during root development. The results obtained using 3D models verified that the continuous HERS sheet acts as a guide for root canal segregation and, after disintegration with the underlying dentin formation, HERS-derived epithelial cells have diverse fates, including migration into the periodontium and embedding in the cementum. Recent advances in 3D imaging technology allow us to revisit multiple key issues regarding the developmental morphogenesis and pathological entities of teeth and related tissues.

Use of Microfocus X-ray Computer Tomography for 3D-image Construction and Quantitative Morphoanalysis

Abstract Microfocus X-ray computed tomography (μCT) is becoming widely used in various research fields relevant to the oral sciences. This technology, in conjunction with computer-assisted 3D reconstruction and quantitative structural analysis, is most suitable for investigating hard tissues in a non-invasive way. Despite this recognition, it still remains a concern whether the data acquisition process is conducted properly and accurately in order to ensure the reproducibility of 3D imaging and the determination of structural parameters such as bone volume (BV) and surface area (BS). In this report, we aim to provide an overview of the experimental procedures and conditions that are necessary for optimizing the acquisition of gray-scale CT images and their conversion into digital images (TIFF or JPEG) prior to 3D reconstruction and measurements. Towards these objectives, we collected μCT images of a 4-week-old mouse temporomandibular joint using a μCT apparatus (Nittetsu ELEX Co., ELE-SCAN model). Data proceedings and measurements were conducted using software (RATOC System Engineering Co., TRI-BON and TRI-SRF2). Special care was taken in optimizing experimental conditions and variables, e.g., X-ray tube voltage and current, selection of an adequate shiftvalue to compensate the mismatching between the specimen-rotating center and incident X-ray beam, and window level and width for contrast adjustment. The results of our empirical approach showed that careful selection of the experimental conditions and computation variables ensures the high quality of the 3D images and the accuracy and reproducibility of the quantitative measurements. The magnitude of errors associated with each of the determined structural parameters was confirmed to be within 3% of the determinations. Further knowledge and refinement of μCT technology and data acquisition will help to improve our understanding of the architecture and dynamic modeling of hard tissues.

Three-dimensional visualization of perlecan-rich neoplastic stroma induced concurrently with the invasion of oral squamous cell carcinoma

BACKGROUND We have demonstrated the induction of perlecan-rich stroma of oral squamous cell carcinoma (SCC) on and after its start of invasion. However, it remains unknown how such a neoplastic stroma is actually arranged in tumor tissues. METHODS To this end, tissue microarray samples, in which keratin and perlecan were contrastively labeled by immunohistochemistry, were three-dimensionally analyzed using digital images and image analysis software to demonstrate the relationship between SCC foci and the perlecan-positive stromal space or that between carcinoma in situ (CIS) and invasive SCC foci. RESULTS The three-dimensional (3D) reconstruction demonstrated three kinds of perlecan profiles for inside (I) and outside (O) areas of the carcinoma cell focus: mode 1, I(+)/O(-) ; mode 2, I(+)/O(+) ; and mode 3, I(-)/O(+). Mode 1 was seen in CIS as well as SCC tumor massifs in the surface part. Mode 2 was seen in small SCC foci, which seemed isolated in 2D sections but were mostly continuous with the tumor massif in 3D reconstructions. Mode 3 was limited to small SCC foci, which were truly segregated from the tumor massif. CONCLUSIONS The results indicated that the 2D SCC focus isolation could not be regarded as invasion but that the SCC foci surrounded by perlecan-positive stroma (modes 2 and 3) could be regarded as a more objective measure for invasion of SCC. This is the first 3D tissue-level demonstration of the neoplastic stroma space induced with oral SCC invasion, the presence of which we have predicted based on our previous 2D and tissue culture evidence.

Modulatory Mechanism of Nociceptive Neuronal Activity by Dietary Constituent Resveratrol

Changes to somatic sensory pathways caused by peripheral tissue, inflammation or injury can result in behavioral hypersensitivity and pathological pain, such as hyperalgesia. Resveratrol, a plant polyphenol found in red wine and various food products, is known to have several beneficial biological actions. Recent reports indicate that resveratrol can modulate neuronal excitability, including nociceptive sensory transmission. As such, it is possible that this dietary constituent could be a complementary alternative medicine (CAM) candidate, specifically a therapeutic agent. The focus of this review is on the mechanisms underlying the modulatory effects of resveratrol on nociceptive neuronal activity associated with pain relief. In addition, we discuss the contribution of resveratrol to the relief of nociceptive and/or pathological pain and its potential role as a functional food and a CAM.