Contents

Review article:

1. Ultrastructural characterization of the interstitial cells of Cajal (KOMURO, T.)

Original articles:

1. Scanning electron microscopic detection of nuclear structures involved in DNA replication (GOBBI, P.)
   
2. Growth of collagen fibrils produced by human osteosarcoma cells: high-resolution scanning electron microscopy (HASHIZUME, H.)
   
3. Regional differences in the cellular proliferation activity of the regenerating rat pancreas after partial pancreatectomy (HAYASHI, K.)
   
4. The serous demilune of rat sublingual gland is an artificial structure produced
   by conventional fixation (YAMASHlNA, S.)
   
5. Dynamics of astrocyte adhesion as analyzed by a combination of atomic force
   microscopy and immunocytochemistry: the involvement of actin filaments and
   connexin 43 in the early stage of adhesion (YAMANE, Y.)
   
6. Heterogeneous reactivity of murine epidermal Langerhans cells after application of FITC: a histochemical evaluation (DOl, S.)
   
7. Three-dimensional architecture of the keratin filaments in epithelial cells surrounding taste buds in the rat circumvallate papilla (SAKAI, H.)
   
8. Cytoplasmic delayed neuronal death in the myenteric plexus of the rat small
   intestine after ischemia (PIAO, D.)
   
9. Succinate dehydrogenase activities of fibers in the rat extensor digitorum
   longus, soleus, and cardiac muscles (NAKATANI, T.)
   

Summary

Title
Ultrastructural Characterization of the Interstitial Cells of Cajal

Author
Terumasa KOMVRO, Keisuke SEKI and Kazuhide HORIGVCHI

Address
Department of Basic Human Sciences, School of Human Sciences, Waseda University, Tokorozawa, Saitama, Japan

Summary. Recent studies on the interstitial cells of Cajal (ICC) have determined ultrastructural criteria for the identification of these previously enigmatic cells. This review deals with the electron microscopic findings obtained by the author's research group in difrerent tissue regions of the gut in mice, rats and guinea.pigs, comparing these with reports from other groups in difrerent species and in humans.

ICC are characterized by the following morphological criteria: numerous mitochondria, abundant intermediate filaments and large gap junctions which connect the cells with each other and with smooth muscle cells. Due to their location in the gut and the specific species, the ICC are markedly heterogeneous in appearance, ranging from cells closely resembling smooth muscle cells to those similar to fibroblasts (Table 1). Nevertheless, the above;mentioned morphological features are shared by all types of ICC and serve in identifying them.

Recent discoveries on a significant role of c.kit in the maturation of the ICC and their specific immuno. reactivity to anti-c-Kit antibody have confirmed the view that the ICC comprise an independent and specific entity of cells. This view is reinforced by the findings of the author's group that the ICC characteristically possess vimentin filaments and are stained with the zinc iodide-osmium tetroxide method which provides a staining aflinity similar to methylene blue, the dye used in the original work by CAJAL (1911). Developmental studies indicate that the ICC are derived from a non. neuronal, mesenchymal origin.

This paper further reviews advances in the physiolo. gical studies on the ICC, in support of the hypothesis by THUNEBERG (1982) that they function as a pacemaker in the digestive tract and a mediator transmitting impulses from the nerve terminals to the smooth muscle cells.

Prof. Terumasa KOMURO
Department of Basic Human Sciences School of Human Sciences
Waseda University
2-579-15 Mikajima, Tokorozawa
Saitama, 359-1164 Japan
Tel: +81-42-947-6730
Fax: +81-42-948-4314
E-mail : komuro@human.waseda ac.jp

Title
Scanning Electron Microscopic Detection of Nuclear Structures Involved in DNA Replication

Author
Pietro GOBBI, Mirella FALCONI, Marco VITALE, Angela GALANZI, Marco ARTICO, Alberto M. MARTELLI and Giovanni MAZZOTTI

Address
Institute of Human Anatomy;, Bologna University Faculty of Medicine, Bologna, Institute of Cytomorphology, CNR, Bologna; Department of Biological Science and Biotechnology, Brescia University Faculty of Medicine, Brescia; Department of Human Anatomy University "La Sapienza" Faculty of Pharmacy, Roma; Department of Human Morphology, Trieste University Faculty of Medicine, Trieste, Italy

Summary. In order to evaluate at the ultrastructural level the three dimensional chromatin arrangement during interphase and particularly during the S phase, the immunogold detection of Bromodeoxyuridine (BrdUI, as a marker of DNA synthesis, was performed in human HeLa, HL60, and in murine Friend leukemia cells (FLC). Field emission in lens scanning electron micros; copy analysis of ultrathin cryosections revealed the presence of a regular three-dimensional network of fibers in dispersed chromatin. This spatial architecture was apparently constituted mainly of 10 nm filaments organized in loops of about 80;l00 nm. Nodal points and the overlapping of such coils appeared as thicker struc. tures of about 30nm in diameter. Thin filaments of ahout 5nm did not show a regular distribution. This three-dimensional fiber organization seemed quite con. stant in the dispersed chromatin of all the cell lines analyzed.

The DNase treatment of the samples selectively removed the 10 nm class fibers, whereas the BrdU labeling confirmed the presence of newly synthesized DNA organized into chromatin units with a regular arrange. ment. These data suggest that the 10 nm chromatin fiber likely represents the DNA condensation order at which DNA duplication starts and the main weft of a three dimensional network within the interphase nucleus.

Prof. Giovanni MAZZOTTI
Institute of Human Anatomy
Bologna University Faculty of Medicine Via Irnerio 48
40126 Bologna Italy
Tel: +39-051-243369
Fax: +39-051-251735
E-mail: mazzotti@biocfarm.unibo.it

Title
Growth of Collagen Fibrils Produced by Human Osteosarcoma Cells: High-Resolution Scanning Electron Microscopy

Author
Hiroya HASHIZUME, Jiro HITOMI and Tatsuo USHIKI

Address
Department of Anatomy and Histology, Niigata University School of Medicine, Niigata, Japan

Summary. To demonstrate three.dimensionally the process of the collagen fibril growth, the bottom of culture dishes with human osteosarcoma cells (NOS.1) and their extracts were examined by conventional scan. ning electron microscopy (SEM). Backscattered elec. tron (BSE) imaging of SEM was also applied to the specimens, which were stained with phosphotungustic acid and uranyl acetate. Conventional SEM images showed several stages of collagen fibril assembly. Short collagen fibrils with tapered ends were distributed at the bottom of the dish just beneath and/or around the cultured cells; they were 1J1m long and 20-30 nm in diameter at the thickest middle portion. These fibrils were often twisted and united in a right helical direc. tion, and consequently increased in length (5-10J1m) and diameter (more than 100 nm). In BSE images, the periodical bands stained with phosphotungstic acid and uranyl acetate were visualized throughout the individ. ual fibrils. The banding pattern indicated that the polarity of the collagen molecules was unidirectional; namely, that all molecules were pointed in the same direction throughout the length of the fibrils.

Dr. Hiroya HASHIZUME
Department of Anatomy and Histology Niigata University School of Medicine Asahimachi-dori, Niigata
951-8510 Japan
Tel: +81-25-227-2059
Fax: +81-25-224-1767
E-mail: hasizume@med.niigata-u.ac.jp

Title
Regional Differences in the Cellular Proliferation Activity of the Regenerating Rat Pancreas after Partial Pancreatectomy

Author
Keiko HAYASHI, Tsuyoshi TAKAHASHI, Akira KAKITA and Shohei YAMASHINA

Address
Departments of Anatomy and Surgery, Kitasato University School of Medicine, Sagamihara, Japan

Summary. The proliferation activity of component cells and its regional dilrerences in the regenerating rat pancreas after 90% pancreatectomy were examined by bromodeoxyuridine (BrdU) immunohistochemistry. Cells of the ductal system and the centroacinar cells showed a rapid increase in labeling indices at day 2 after pancreatectomy, followed by a second peak of a mild increase at days 5 to 7. No regional dilrerence in the labeling index was recognized in the ductal elements. In contrast, the labeling index of acinar cells started to increase at day 3, reaching a definite peak at day 5. Furthermore, acinar cells in the region close to the duodenum had labeling indices more than 2 times higher than those in the portions further away from the duodenum. Acinar cells increased in number as early as from day 3 after surgery. These result suggested that the parental cells of regeneration were located in the ductal epithelium. It is highly probable that the proliferation of acinar cells is controlled by some unknown trophic factor(s) which is released locally from the duodenum, but does not involve a neural or a circulatory route. The phenomenon may be closely linked to the known fact that the incidence of pancreatic cancer is highest in the head region.

Keiko HAYASHI, M. D.
Department of Surgery
Kitasato University School of Medicine 1-15-1, Kitasato, Sagamihara
228-8555 Japan
Tel: +81-42-778-9021
Fax: +81-42-778-9372

Title
The Serous Demilune of Rat Sublingual Gland Is an Artificial Structure Produced by Conventional Fixation

Author
Shohei YAMASHINA, Hideaki TAMAKI and Osamu KATSUMATA

Address
Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Japan

Summary. The ultrastructure of the secretory end. piece of the rat sublingual gland was examined in samples prepared by rapid freezing and freeze.substitution method, and results were analyzed in combination with 3-D images reconstructed by computer graphics from light micrographs of serial sections. Fixation by rapid freezing followed by freeze.substitution preserved cellular ultrastructures, especially the membrane structure, in perfect condition, and demonstrated the termi. na! portion of the sublingual gland to be a compound branched tubulo..alveolar gland with serous cells dis. tributed throughout the end.pieces. All the serous cells aligned with mucous cells to surround a common lumen, leaving no demilune structure. In contrast, samples fixed by the conventional immersion method showed distended mucous cells displacing the serous cells toward the basal portion of the acinus to form the demilune structure. The lumina! space was also compressed and appeared disconnected from the serous cells. From these observations, the serous demilune that for more than 130 years has been believed to be an actual histological entity was proved to be an artificial structure produced through compression by the hydrated and expanded mucous cells during immersion fixation.

Prof. Shohei YAMASHINA, M.D.
Department of Anatomy
Kitasato University School of Medicine 1-15-1, Kitasato, Sagamihara
228-8555 Japan
Tel: +81-42-778-8826
Fax: +81-42-778-8441
E-mail: yamasina@kitasato-u.ac.jp

Title
Dynamics of Astrocyte Adhesion as Analyzed by a Combination of Atomic Force Microscopy and Immunocytochemistry: the Involvement of Actin Filaments and Connexin 43 in the Early Stage of Adhesion

Author
Yukako YAMANE, Hatsuki SHIGA, Hiroaki ASOU, Hisashi HAGA, Kazushige KAWABATA, Kazuhiro ABE and Etsuro ITO

Address
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo; Department of Neuro-Cell Biology, Tokyo Metropolitan Institute of Gerontology, Tokyo; Division of Physics, Graduate School of Science, Hokkaido University, Sapporo, and Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan

Summary. We observed the time-dependent morphological alteration of astrocytes during their adhesion by atomic force microscopy (AFM) and investigated the relationships between this morphological alteration and the localization of actin filaments and connexin 43 by immunocytochemistry. The fine processes observed as fine ridge-like structures by AFM were closely concerned with actin filaments by immunocytochemistry. During the adhesion of astrocytes, actin filaments appeared to be aligned regularly beyond the borders among dilrerent cells. Detectable connexin immunoreactivity was changed in the following regions: 1) the tips of fine cell processes and the cell margin when astrocytes started to adhere; 2) the border of cells when astrocytes tightly adhered; and 3) non-specific sites when astrocytes became a cluster. In the former two cases, the immunopositive spots for connexin were observed to colocalize with the tips of cell processes with actin filaments. These results strongly suggest that connexin associated with actin filaments at the tip of cell processes plays an important role in the early stage of the adhes ion of astrocytes. These observations alrord valuable clues for understanding the glial communication.

Dr. Etsuro ITO
Division of Biological Sciences Graduate School of Science
Hokkaido University
North 10, West 8, Kita-ku
Sapporo, 060-0810 Japan
Tel: +81-11-706-2615
Fax: +81-11-706-4448
E-mail : eito@sci.hokudai.ac.jp

Title
Heterogeneous Reactivity of Murine Epidermal Langerhans Cells after Application of FITC: a Histochemical Evaluation

Author
Satoru DOI, Miya KOBAYASHI, Yasuo SUGIURA, Tatsuo SAKAMOTO and Shinpei TORII

Address
Department of pediatrics and Anatomy II, Nagoya University School of Medicine, Nagoya, Japan

Summary. To elucidate the detailed kinetics of epider. ma! Langerhans cells after topical contact sensitizer stimulation, we examined ATPase or Ia positive epider. ma! cells of BALB/c mice in a time.spaced manner after the topical application of fluorescein isothiocyanate (FITC). We also performed double labeling of Langer. hans cells in epidermal sheets with ATPase activity and Ia antigen or costimulatory molecules (B7.1 and B7.2) after the same stimulation. Observations showed that the density of ATPase positive cells and Ia positive cells decreased following a dilrerent time course; the former reached a nadir (77.4% of control) at 4 h but the latter reached a minimum (82.8% of control) at 16 h after the application of FITC. A double labeling technique re. vealed an increase in Ia single positive cells at 4 h as opposed to that of ATPase single positive cells at 16 h after application. Both costimulatory molecules were expressed on the dendritic processes of many Langer. hans cells as a dotty pattern at 4 h after application ; B7 positive and ATPase negative areas were observed at this time. On electron microscopic observation, a few activated Langerhans cells found in the dermis at 4 h after application had distinctive profiles compared with residual Langerhans cells in the epidermis. These findings suggest that there is a heterogeneity of rectivity to FITC in epidermal Langerhans cells, and that only a small portion of them migrates from the epidermis during sensitization. The findings also indi. cate the importance of the interaction between the Langerhans cell and its surrounding microenvironment in the epidermis for its activation. In addition, the results indicate that the enzymatic and the phenotypic markers do not definitively reflect the persence (or absence) of Langerhans cells.

Dr. Satoru DOI
Department of Pediatrics
Hekinan Municipal Hospital
3-6, Heiwa-machi, Hekinan-city 447-8502 Japan
Tel: + 81-566-48-5050
Fax: + 81-566-48-7522

Title
Three-dimensional Architecture of the Keratin Filaments in Epithelial Cells Surrounding Taste Buds in the Rat circumvanate Papilla

Author
Hiroatsu SAKAI, Toshiyuki KAIDOH, Shinichi MORINO and Takao INOUE!

Address
Department of Anatomy and Laboratory of Electron Microscopy, Faculty of Medicine, Tottori University, Yonago, Japan

Summary. The three-dimensional architectures of the perigemmal cells and their keratin bundles in the rat circumvallate papillae were studied by transmission and scanning electron microscopy. The perigemmal cells were classified into three layers: basal, middle and upper. The basal layer consisted of polygonal cells located close to the basal lamina, the middle layer comprised longitudinally elongated cells fitting the lateral convexity of the taste bud, and the upper layer was imbricating flat cells along the upper portion of the taste bud. When fresh specimens were jointly treated with Triton X-100 and sonication, the taste buds were often detached and the cytoplasmic matrices of the perigemmal cells were elrectively removed. Consequently, we were able to demonstrate an extensive network of the subplasmalemmal keratin bundles of the perigemmal cells. The framework appeared either as a thin lacework, a thick fence-like structure, or a lattice work in the basal, middle, and upper layers, respectively. The thin lacework in the basal layer was considered to be a developing process of the framework. The thick fence-like structure in the middle layer probably plays a primary role in supporting the taste bud. The latticework in the upper layer is believed to reflect a remodeling in reducing the keratin framework.

Dr. Hiroatsu SAKAI
Departmeut of Anatomy Faculty of Medicine
Tottori University
Yonago, Tottori
683-8503 Japan
Tel: +81-859-34-8004
Fax: +81-859-34-8167

Title
Cytoplasmic Delayed Neuronal Death in the Myenteric Plexus of the Rat Small Intestine after Ischemia

Author
Da Xun PIAO, Hong Chi JIANG, Motoyasu KOSAKA, Takashi SHIBATA, Aiji OHTSUKA and Takuro MURAKAMI

Address
Department of Anatomy, Okayama University School of Medicine, Okayama, Japan; Department of Surgery, First Hospital, Harbin Medical University, Harbin, China; and Terminal Care Research Center, Shibata Hospital, Kurashiki, Japan

Summary. The present study demonstrates light and electron microscopic changes in neurons in the myenteric plexus of the rat ileum following four.hour ischemia. Macroscopically, an intestinal constriction occurred at the damaged portion at three weeks after ischemia ; the segment oral to the constriction markedly swelled at four weeks. In light microscopy, at three weeks after ischemia, the myenteric neurons appeared spongy or foamy, containing many vacuoles in their somatic cytoplasm. At four weeks, the neuronal cytoplasm and nerve fiber bundles had disintegrated to form vacant spaces in the myenteric plexus. The neuronal nucleus of the damaged plexus did not show positive nick.end labeling. In electron microscopy, neuronal cytoplasm revealed degenerative signs already at one week after ischemia : a distended endoplasmic reticulum and swollen mitochondria with fragmentary cristae. The nerve fibers also showed destruction of the mitochondria, and degenerative changes in the postsynaptic sites appeared earlier than the presynaptic terminals. The results suggest that intestinal ischemia causes delayed neuronal death, which diners from the apoptotic process previously demonstrated in the ischemia.damaged brain.

Dr. Aiji OHTSUKA
Department of Anatomy
Faculty of Medicine
Okayama University Medical School 2-5-1 Shikata-cho, Okayama
700-8558 Japan
Tel: +81-86-235-7088
Fax: +81-86-235-7095
E-mail: em2kai@med.okayama-u.ac.jp

Title
Succinate Dehydrogenase Activities of Fibers in the Rat Extensor Digitorum Longus, Soleus, and Cardiac Muscles

Author
Toshiaki NAKATANI, Toshikatsu NAKASHIMA, Taizo KlTA, Chiyoko HIROFUJI, Kazuo ITOH, Minoru ITOH and Akihiko ISHIHARA

Address
Department of Pharmacology, Nara Medical University, Nara; Human Performance and Exercise Prescription Laboratory Tenri University, Tenri; Laboratory of Exercise Physiology, Osaka Gakuin University, Suita; Graduate School of Integrated Science and Art University of East Asia, Shimonoseki; Prof. Emeritus, Kyoto University, Kyoto; and Laboratory of Neurochemistry, Facuity of Integrated Human Studies, Kyoto University, Kyoto, Japan

Summary. Succinate dehydrogenase (SDH) activities and cross.sectional areas (CSAs) of digerent types of fibers in the superficial (EDLs) and deep (EDLd) regions of the extensor digitorum longus and soleus (SOL) muscles and the left ventricular muscle of the heart (HEART) of 10-week-old male rats were determined using quantitative histochemistry and a computerassisted image processing system. The fibers were classified as type I, type IIA, type IIB, or type IIC according to their histochemically assessed adenosine triphosphatase activities. The mean SDH activity was higher and the mean CSA was smaller in type IIA fibers than in type IIB fibers in both the EDLs and EDLd. The mean SDH activity of type IIA fibers in the SOL was higher than that of type I fibers. Fibers in the HEART showed the highest mean SDH activity and the smallest mean CSA among all fiber types in the muscles examined. There was an inverse correlation between CSA and SDH activity for the digerent fiber types in digerent muscles. These data suggest that the SDH activity of fibers in muscle is fiber type- and size-specific, and that the highest SDH activity of fibers in the left ventricular muscle of the heart contributes to their functional properties, i.e., high fatigue resistance.

Dr. Akihiko ISHIHARA
Laboratory of Neurochemistry
Faculty of Integrated Human Studies Kyoto University
Sakyo-ku, Kyoto
606-8501 Japan
Tel: +81-75-753-6881
Fax: +81-75-753-6771
E-mail: ishihara@life.h.kyoto-u.ac.jp