Contents

Review article

1. Morphological and cytochemical characteristics of periodontal Ruffini ending under normal and regeneration processes (WAKISAKA, S.)

Original articles

1. Scanning electron microscopic studies of the vascular smooth muscle cells and pericytes in the rat heart (HIGUCHI, K.)

2. The suhfibrillar arrangement of corneal and scleral chollagen fibrils as revealed by scanning electron and atomic force microscopy (YAMAMOTO, S.)

3. Histometrical and three-dimensional analyses of liver hematopoiesis in the mouse embryo (SASAKI, K.)

4. Semiquantitative morphological analysis of stromal cells in the irradiated and recovering rat thymus (ARUDCHELVAN, Y.)

5. Phenotypical and morphological analyses of intraepithelial and lamina propria lymphocytes in normal and regenerating gastric mucosa of rats in comparison with those in intestinal mucosa (NAKAGAWA, K.)

6. Time-related changes of developing enamel crystals after exposure to the tissue fluid in vivo(i) : analysis of a subcutaneously implanted rat incisor (YAZAWA, H.)

7. Villiform processes in the pharynx of the soft-shelled turtle, Trionyx sincnsis japoncus(i), functioning as a respiratory and presumably salt uptaking organ in the water (YOKOSUKA, H.)

8. The vascular supply of the villiform processes in the pharynx of the soft-shelled turtle, Trionyx sincnsis japonicus(i). A scanning electron microscopic study of corrosion casts (YOKOSUKA H.)

Summary

Title
Morphological and Cytochemical Characteristics of Periodontal Ruffini Ending under Normal and Regeneration Processes

Author
Satoshi WAKISAKA, Yukako ATSUMI, Suk Hyun YOUN and Takeyasu MAEDA

Address
Department of Oral Anatomy and Developmental Biology Osaka University Faculty of Dentistry, Osaka ; and Department of Oral Anatomy, Faculty of Dentistry, Niigata University, Niigata, Japan

Summary
Current knowledge on the Ruffini endings, primary mechanoreceptors in the periodontal ligament is reviewed with special reference to their cytochemical features and regeneration process. Morphologically, they are characterized by extensive ramifications of expanded axonal terminals and an association with specialized Schwann cells, called lamellar or terminal Schwann cells, which are categorized, based on their histochemical properties, as non-myelin-forming Schwann cells. Following nerve injury, the periodontal Ruffini endings of the rat incisor ligament can regenerate more rapidly than Ruffini endings in other tissues. During regeneration, terminal Schwann cells associated with the periodontal Ruffini endings migrate into regions where they are never found under normal conditions. Also during regeneration, alterations in the expression level of various bioactive substances occur in both axonal and Schwann cell elements in the periodontal Ruffini endings. Neuropeptide Y, which is not detected in intact periodontal Ruffini endings, is transiently expressed in their regenerating axons. Growth-associated protein-43 (GAP-431 is expressed transiently in both axonal and Schwann cell elements during regeneration, while this protein is localized in the Schwann sheath of periodontal Ruffini endings under normal conditions. The expression of calbindin D28k and calretinin, both belonging to the bufrering type of calcium-binding proteins, was delayed in periodontal Ruffini endings, compared to their morphological regeneration. As the importance of axon-Schwann cell interactions has been proposed, further investigations are needed to elucidate their molecular mechanism-particularly the contribution of growth factors-during the regeneration as well as development of the periodontal Ruffini endings.

Dr Satoshi WAKISAKA
Department of Oral Anatomy and
Developmental Biology
Osaka University Faculty of Dentistry 1-8, Yamadaoka, Suita, Osaka
565-0871 Japan
Tel: +81-6-6879-2872
Fax: +81-6-6879-2875
E-mail: wakisaka@dent.osaka-u.ac.jp

Title
Scanning Electron Microscopic Studies of the Vascular Smooth Muscle Cells and Pericytes in the Rat Heart

Author
Kotaro HIGUCHI, Hiroya HASHIZUME, Yoshifusa AIZAWA and Tatsuo USHIKI

Address
Departments of Anatomy and Histology, and Internal Medicine, Faculty of Medicine, Niigata University, Niigata, Japan

Summary
The cytoarchitecture of smooth muscle cells and pericytes in the rat cardiac vessels was studied by scanning electron microscopy after the removal of connective tissue matrices using a modified KOH collagenase digestion method. The initial stem of the coronary arteries had groups of smooth muscle cells which ran in various directions on the outermost layer of the media. Although smooth muscle cells in coronary arteries of more than 100 Jim in the outer diameter were arranged in a rough circle around the vessel axis, oblique and/or longitudinal muscle bundles were often present in the medio-adventitial border of the vessels. The presence of irregularly oriented muscular bundles is probably connected with resistance against the stretching force induced by the beating of the heart. As the vessel size decreased toward the periphery, almost all of the smooth muscle cells became spindle-shaped with several tiny processes and ran circularly or helicaly to the vessel axis. In the precapillary arterioles (6-12 Jim), smooth muscle cells acquired various cytoplasmic processes which helicaly surrounded endothelial cells. Unmyelinated nerves were often associated with arterioles. Blood capillaries were morphologically divided into three segments: arterial capillaries which had pericytes with wide and circularly oriented processes, true capillaries whose pericytes extended long and thin primary processes bilaterally along the vessel axis, and venous capillaries surrounded irregularly and loosely by wide pericytic processes. The stellate pericytes in the postcapillary venules (10l30 Jim) gradually changed into flat tape-like smooth muscle cells, which ran circularly in the collecting venules and veins (30-200 Jim). The large collecting veins were finally overwhelmed by superficial thin layer of the myocardium, their own smooth muscle cells being very sparse. This suggests that large veins have poor ability to contract by themselves but are influenced by the surrounding myocardial cells

Dr. Kotaro HIGUCHI
First Department of Internal Medicine Faculty of Medicine
Niigata University
Asahimachi-dori 1, Niigata
951-8510 Japan
E-mail (to Prof. USHIKI):
t-ushiki@med.niigata-u.ac.jp

Title
The Subfibrillar Arrangement of Corneal and Scleral Collagen Fibrils as Revealed by Scanning Electron and Atomic Force Microscopy

Author
Susumu YAMAMOTO, Hiroya HASHIZUME, Jiro HITOMIl, Masatsugu SHIGENO, Shoiehi SAWAGUCHI, Haruki ABE and Tatsuo USHIKI

Address Departments of Anatomy and Histology and of Ophthalmology, Faculty of Medicine, Niigata University, Niigata; Seiko Instruments Inc., Chiba; and Department of Ophthalmology, Ryukyu University School of Medicine, Okinawa, Japan

Summary
The present study was designed to analyze the subfibrillar structure of cornea! and sclera! collagen fibrils by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Isolated collagen fibrils of the bovine cornea and sclera were fixed with l% OsO4, stained with phosphotungstic acid and uranyl acetate, dehydrated in ethanol, critical point-dried, metal-coated, and observed in an in-lens type field emission SEM. Some isolated collagen fibrils were fixed with l% OsO4, dehydrated, critical point-dried and observed without metal-coating in an AFM. Isolated collagen fibrils treated with acetic acid were also examined by SEM and AFM. SEM and AFM images revealed that cornea! and sclera! collagen fibrils had periodic transverse grooves and ridges on their surface; the periodicity (i. e., D-periodicity) was about 63 nm in the cornea and about 67 nm in the sclera. Both cornea! and sclera! collagen fibrils contained subfibrils running helicoidally in a rightward direction to the longitudinal axis of the fibril; the inclination angle was about 15o in the cornea! fibrils and So in the sclera! fibrils. These findings indicate that the different D-periodicity between corneal and scleral fibrils depends on the different inclinations of the subfibrils in each fibril. The present study thus showed that cornea! collagen fibrils differ from sclera! collagen fibrils not only in diameter but also in substructure.

Dr. Susumu YAMAMOTO
Department of Ophthalmology Faculty of Medicine
Niigata University
Asahimachi-dori, Niigata
951-8510 Japan
Tel: +81-25-227-2296
Fax: +81-25-227-0785
E-mail: shin@med.niigata-u.ac.jp

Title
Histometrical and Three-Dimensional Analyses of Liver Hematopoiesis in the Mouse Embryo

Author
Kazunobu SASAKI and Yuji SONODA

Address
Department of Anatomy, Kawasaki Medical School, Kurashiki, Japan

Summary
The development and cytoarchitectures of liver hematopoiesis in the mouse from 10 to 19 days of gestation were examined by light and electron micros; copy. In fetal liver hematopoiesis, four stages were identified: Stage I, the onset of hematopoiesis at 10 days; Stage II, expansion of the volume of the hematopoietic compartment at 11 and l2days; Stage III, the peak in the volume of the hematopoietic compartment at 13 and 14days; and stage IV, the involution of hematopoiesis after 15 days. During stages I-II, hematopoietic stem cells appeared to move from the sinusoidal lumina into primitive hepatic cell cords through the sinusoidal endothelium to give rise to colonies among hepatoblasts. At Stage III, the hematopoietic colonies formed ellipsoidal foci as a structural unit of hematopoiesis. These foci were 35-70 x 20-40 (micro)m in size, and erythroblastic islands could be observed in the center of each. Each island contained central macrophages surrounded by a ring of erythroblasts. The macrophages underwent mitosis, showing close contact with the erythroblasts, after which the hematopoietic foci appeared as cords. At Stage IV, these cord-shaped hematopoietic foci became disrupjed, and round solitary foci including macrophages appeared within the hepatic cell cords on meandering sinusoids. In fetal liver hematopoiesis, macrophages could be one of the major cell components comprising the hemato; poietic microenvironment, especially at Stages II and III.

Prof. Kazunobu SASAKI
Department of Anatomy
Kawasaki Medical School
577 Matsushima, Kurashiki
701-0192 Japan
Tel & Fax: +81-86-462-1111
E-mail: kazus@med.kawasaki-m.ac.jp

Title
Semiquantitative Morphological Analysis of Stromal Cells in the Irradiated and Recovering Rat Thymus

Author
Yamini ARUDCHELVAN, Nobuko TOKUDA, Masakatsu TAMECHIKA2. Yu-Hsueh WANG, Noriko MIZUTANI, Tomoo SAWADA, Kazuhito YAMAGUCHI, Tetsuo FUKUMOTO and
Fumihiko SHINOZAKI

Address
Departments of Oral and Maxillofacial Surgery, Anatomy and Institute of Laboratory Animals, Yamaguchi University School of Medicine, Ube, Japan

Summary
To understand the roles of thymic stromal cells in T.lymphocyte development, we semiquantita. tively analysed rat thymi recovering from irradiation (6 Gy), using a transmission electron microscope. The most striking findings were that the percentage of subcapsular epithelial cells significantly increased in the cortex on day 3 after irradiation compared with the control; the percentage of intermediate epithelial cells significantly increased in the cortex on days 3 and 5 after irradiation and in the medulla on days 5 and 7 compared with the control; the interdigitating cells disappeared from the medulla by day 7 after irradiation and reappeared on day 9. The present data thus reveal that during recovery after irradiation 16 Gy), marked changes occur in the relative proportions of digerent epithelial cell subtypes in the cortex and medulla of the rat thymus. In addition, the percentages of macro. phages and interdigitating cells also changed during the recovery. These changes, which may be associated with the abrupt proliferation of thymocytes after irradiation, should shed light on the significance of stromal cells in the T cell development.

Dr. (Mrs.) Yamini ARUDCHELVAN
Department of Oral and Maxillofacial Surgery
Yamaguchi University School of Medicine
1144 Kogushi, Ube
755-8505 Japan

Title
Phenotypical and Morphological Analyses of Intraepithelial and Lamina Propria Lymphocytes in Normal and Regenerating Gastric Mucosa of Rats in Comparison with Those in Intestinal Mucosa

Author
Koichiro NAKAGAWA, Kazuhide HIGUCHI, Tetsuo ARAKAWA, Kenzo KOBAYASHI and Kenji KANEDA

Address
Departments of Anatomy and Internal Medicine, Osaka City University Medical School, Osaka, Japan

Summary
While the intestine has abundant intraepithelial lymphocytes (IELs) including extrathymically differentiated T-cell populations and natural killer (NK) cells, the stomach contains only a few IELs. To elucidate whether the gastric epithelium is capable of inducing predominant lymphocyte lodging and subsequent differentiation within, we counted the number of IELs and lamina propria lymphocytes (LPLs) and calculated the percentage of IELs to total lymphocytes for each alpha-beta (ab) T cell, gamma-dalta (cd) T cell, CD4+ cell, CD8+ cell and NK cell in normal and regenerating gastric mucosa as well as the intestinal mucosa of the rat. In the normal rat pylorus, a few abT cells but no cd cells were found in the epithelium and lamina propria. In regenerating gastric mucosa, all subsets of LPLs increased in number to a degree comparable to those in intestinal mucosa, whereas every IEL subset, though slightly increased, was much smaller in number than in the intestinal mucosa, consequently giving lower percentages of IELs. Electron microscopic observations revealed that all IELs in regenerating gastric mucosa were agranular, while 55% of intestinal IELs were large granular lymphocytes positively stained for an NK-cell, ab cell or cd T-cell marker. The present results indicate that, unlike the intestinal epithelium, the gastric epithelium does not induce the preferential localization of T cells/NK cells and T-cell differentiation into granular lymphocytes in the epithelium even under conditions of prominent LPL infiltration.

Prof. Kenji KANEDA, M.D.
Department of Anatomy
Osaka City University Medical School 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585 Japan
Tel: +81-6-6645-3705
Fax: +81-6-6646-3603
E-mail : kkaneda@med.osaka-cu.ac.jp

Title
Time-Related Changes of Developing Enamel Crystals after Exposure to the Tissue Fluid in vivo: Analysis of a Subcutaneously Implanted Rat Incisor

Author
Hiroshi YAZAWA, Yoshiro TAKANO and Isao ISHIKAWA

Address
Department of Periodontology and Second Department of Oral Anatomy, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan

Summary
To investigate the elrects of tissue fluid on the growth of enamel crystals, upper and lower incisors extracted from 3.week.old Wistar rats were removed of the enamel organ, implanted subcutaneously in the dorsal portion of animals from the same litter, and harvested at 72 h or 1week after implantation. The grafts were chemically fixed with surrounding tissues and prepared for light and electron microscopy, X.ray microanalysis, or for the immunohistochemistry of amelogenin. Mineralization of implanted enamel layers was examined by contact X-ray microradiography.
The immunoreactivities for 25 kD amelogenin in immature enamel decreased sequentially, starting from the surface to the deeper layers; by 1 week after implantation, no positive reactivities remained in the entire enamel layers at the stages of matrix formation and early maturation. In accordance with the loss of enamel proteins, immature enamel gained mineral den. sity until it attained higher radio opacity than that of the adjacent dentin by 1 week. In contrast, the radio opacity of the full thickness of the enamel at early maturation remained low except for a surperficial thin layer.
Electron microscopy revealed no sign of growth of original enamel crystals, but showed heavy precipitation of electron dense fine granules of calcium phosphate in all layers of the secretory enamel and the superficial layer of enamel at early maturation, which showed high radio opacity. The Ca/P ratio and electron diffraction patterns of the granular materials precipitated between intrinsic enamel crystals indicated the property of hydroxyapatite or octacalcium phosphate though a characteristic ribbon-like profile of enamel crystals was lacking.
These data indicate that the enamel organ blocks exogenous mineral precipitates in growing enamel during the stage of matrix formation and plays an essential regulatory role for fine enamel crystallites to grow into large hexagonal crystals.

Dr. Hiroshi YAZAWA
Department of Periodontology
Faculty of Dentistry
Tokyo Medical and Dental University 
1-5-45 Yushima, Bunkyo-ku, Tokyo 
113-8549 Japan
E-mail : yazawa.peri@dent.tmd.ac.jp

Title
Villiform Processes in the Pharynx of the Soft-Shelled Turtle, Trionyx sinensis japonicus, Functioning as a Respiratory and Presumably Salt Uptaking Organ in the Water

Author
Hiroyuki YOKOSUKA, Mikio ISHIYAMA, Sumio YOSHIE and Tsuneo FUJITA

Address
Department of Histology, Nippon Dental University School of Dentistry at Niigata, Niigata, Japan

Summary
Some species of soft-shelled turtle have been known to use a conspicuous mass of villiform processes of the pharyngeal mucosa as an aquatic respiratory organ when staying underwater for prolonged periods, such as hibernation. Using hibernating turtles, Trionyx sinensis japonicus, the present study employed scanning electron microscopy to demonstrate for the first time the detailed morphology and distribution of these villiform processes. Two types of processes, complex and simple, could be identifited.
Light microscope observation of the transverse sections of the villi demonstrated a rich vascularization in the connective tissue of the villi, comprising arterioles and venules running in the core and capillaries in the periphery. Most of the capillaries were invaginated into the multilayered cuboidal epithelium. Near the tip of the villi they became swollen, forming sinusoidal capillaries.
Transmission electron microscopy clarified the fine structure of the blood-water barrier, which consisted of a non-fenestrated endothelium and an attenuated epithelium that sandwiched a connective tissue with a discontinuous subendothelial and a continuous subepithelial basement lamina. The epithelium consisted of secretory cells, mitochondria-rich cells, and basal cells.
The mitochondria-rich cells contained a cytoplasmic area filled with tubulovesicular elements. Based on their ultrastructural resemblance with the chloride cells in the fish and tadpole, these cells are suggested to be involved in the uptake of Na+ and Cl- from fresh water for keeping ionic balance in the blood.

Dr. Hiroyuki YOKOSUKA
Department of Histology
Nippon Dental University
School of Dentistry at Niigata 1-8 Hamauracho, Niigata
951-8580 Japan
Tel: +81-25-267-1500
Fax: +81-25-267-1134
E-mail: yokosuka@ngt.ndu.ac.jp

Title
The Vascular Supply of the Villiform Processes in the Pharynx of the Soft -Shelled Turtle, Trionyx sinensis japonicus. A Scanning Electron Microscopic Study of Corrosion Casts

Author
Hiroyuki YOKOSUKA, Takuro MURAKAMI, Mikio ISHIYAMA, Sumio YOSHIE and Tsuneo FUJITA

Address
Department of Histology, Nippon Dental University School of Dentistry at Niigata, Niigata; and Department of Anatomy, Okayama University School of Medicine, Okayama, Japan

Summary
Following our observations of the fine structure of the pharyngeal villiform processes of the hibernatin g soft - shelled turtle, Trionyx sinensis japonicus (YOKOSUKA et el., 2000), this paper deals with a scanning electron microscope study of the resin casts of blood vessels supplying those processes.
Each villiform process contained arterioles and venules which ran in the axial portion of the process; capillaries formed a network at the periphery of the connective tissue core of the villus. In the distal portions of the villus, the capillaries increased markedly in their caliber to form sinusoidal capillaries. Such a vascular architecture supports the view that the villiform processes serve in the aquatic respiration of the soft-shelled turtle.
The casts indicated an occurrence of sphincters in the vascular bed of the villi.

Dr. Hiroyuki YOKOSUKA
Department of Histology
Nippon Dental University
School of Dentistry at Niigata 1-8 Hamauracho, Niigata
951-8580 Japan
Tel: +81-25-267-l500
Fax: +81-25-267-1134
E-mail: yokosuka@ngt.ndu.ac.jp