Contents

Original articles

1. Immunohistochemical demonstration of nerve terminals in the whole hard palate of rats by use of an antiserum against protein gene product 9.5 (PGP 9.5) (MITSUI, C.)
   
2. The cytoarchitecture of the adult human parabrachial nucleus: a Nissl and Golgi study (GIOIA, M)
   
3. A corrosion casting/scanning electron microscope method that simultaneously demonstrates clear outlines of endothelial cells and three-dimensional vascular organization (OHTANI, O.)
   
4. The presence of a local immune system in the upper blind and lower part of the human nasolacrimal duct (SIRIGU, P.)
   
5. Surface remodeling associated with vasopressin-induced membrane traffic in L6 myogenic cells (COLETTI, D.)
   
6. The ultrastructure of skeletal and smooth muscle in experimental protein malnutrition in rats fed a low protein diet (OUMI, M)
   
7. Enhanced visualization of weak-colloidal iron signals with Bodian's protein silver for demonstration of perineuronal nets of proteoglycans in the central nervous system (HONG, L. J.)
   
8. Morphology of the glomerular nerve endings in the dorsal nasal ligament of the dog (YAMAMOTO, Y.)
   
9. Kupffer cell activation and hematopoiesis in the liver of autoimmune MRL-lpr/lpr mice (CHO, K.)
   
10. Intensely negative-charged pericapillary spaces in the rat pineal gland (TAGUCHI, T.)
    
11. Effects of short-term denervation and subsequent reinnervation on motor end-plates and the soleus muscle in the rat (SAKAKIMA, H.)

Summary

Title
Immunohistochemical Demonstration of Nerve Terminals in the Whole Hard Palate of Rats by Use of an Antiserum against Protein Gene Product 9.5 (PGP 9.5)

Author
Chihiro MITSUI, Toshihiko IWANAGA, Shigemitsu YOSHlDA and Takao KAWASAKI

Address
Department of Oral Functional Science, Graduate School of Dentistry; and Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Summary
Sensory innervation of the entire hard palate was investigated in the rat using serial sections immunostained for protein gene product 9.5 (PGP 9.5), a neuronal marker. PGP 9.5-immunoreactive nerve endings were widely distributed in the hard palate, but the innervation pattern and density differed among portions. They were numerous at papillary protrusions including the incisal papilla, antemolar/intermolar rugae, and postrugal filiform papillae. Immunoreactive free nerve endings gathered at the summits of the connective tissue papillae, some of them entering deeply into the epithelium. Electron microscopy demonstrated that nerves in the postrugal filiform papillae reached the stratum corneum. The atrial region, possibly the most sensitive in the hard palate, showed unique innervation: its anterior part, adjacent to incisors, developed intraepithelial networks of fine and beaded nerves, whereas its posterior part revealed cone-shaped nerve terminals formed on the connective tissue papillae of the atrial folds which comprised two lines of longitudinal flaps. Taste bud-like corpuscles gathered in the medial walls of the incisal canals and in the "Geschmacksstreifen" (taste stripes) present at the most anterior part of the soft palate. The hard palate of the rat is thus richly innervated, and is characterized by region-specific nerve endings which may be involved in mechano- and chemoreception in the oral cavity.

Dr. Chihiro MITSUI
Department of Oral Function
Graduate School of Dentistry
Hokkaido University
Kita 13 Nishi 7, Kita-ku
Sapporo, 060-8586 Japan
Tel: +81-11-706-4270
E-mail: mitsui@den.hokudai.ac.jp

Title
The Cytoarchitecture of the Adult Human Parabrachial Nucleus: a Nissl and Golgi Study

Author
Magda GIOIA, Luigi RODELLA, Maria Grazia PETRUCCIOLI and Rossella BIANCHI

Address
Institute of Human Anatomy, University of Milan; and Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy

Summary
The parabrachial nucleus (PBN) plays important roles in numerous autonomic functions and in pain modulation. In different animal species, three main regions of the PBN have been identified: the m-PB, the l-PB, and the Kolliker-Fuse nucleus (KF). The KF has not been identified in humans. The present study used Nissl and Golgi-Cox material and morphoquantitative methods to investigate the cytoarchitectural organization of the adult human PBN, paying particular attention to neuronal features endowed with functional significance, i. e. the arborization of the neurons. The PBN neuron population is made up of elements which are heterogeneous in size, shape and dendritic arborization, and grouped into two regions, the lateral and medial PBN (l- and m-PB). It has been suggested that some large sized neurons located in the ventral region of the m-PB might be the counterpart of the KF. In the m-PB the fusiform neurons are the most numerous cells; in the l-PB the multipolar neurons prevail, and are particularly numerous in the dorsal l-PB. Since the dendritic arborization is generally the main target of afferent projections to a neuron, it is possible that the l-PB, and in particular its dorsal region, might be the main site for the endings of afferences to the human PBN.

Prof. Magda GIOIA
Istituto di Anatomia Umana
Via Mangiagalli 31
20133 Milano, Italy
Tel: +39 02 70646216
Fax: +39 02 2364082
E-mail: magda.gioia@unimi.it

Title
A Corrosion Casting/Scanning Electron Microscope Method That Simultaneously Demonstrates Clear Outlines of Endothelial Cells and Three-Dimensional Vascular Organization

Author
Osamu OHTANI and Yuko OHTANI

Address
Department of Anatomy, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan

Summary
This paper describes a method that can definitively demonstrate endothelial cell boundaries on corrosion casts of arteries, veins, and capillaries. After perfusion with silver nitrate, a casting medium was injected into the entire vascular bed. The injected tissues were either exposed to light or immersed in the photographic developer to develop the silver halide, and corroded in a 5% NaOH solution at 60 degrees C overnight. Observations of the casts containing water in a low vacuum scanning electron microscope equipped with a cooling stage clearly showed endothelial cell boundaries on casts of every type of vessel as well as their three-dimensional architecture. The low vacuum scanning electron microscope images of wet casts were almost identical in quality to the back-scattered electron images of dried casts without any coating. Secondary electron images of the dried casts with metal coating clearly showed endothelial cell outlines and nuclear imprints. The secondary electron images at high magnification indicated that silver granules were precipitated in the grooves along endothelial cell boundaries on the casts. Since this method can demonstrate endothelial cell boundaries of every type of vessel in addition to their three-dimensional architecture, it will be a powerful tool for examining endothelial cell morphology and microvascular organization in pathological as well as normal tissues.

Prof. Osamu OHTANI
Department of Anatomy
Toyama Medical and Pharmaceutical University
2630 Sugitani, Toyama
930-0194 Japan
Tel: +81-76-434-7205
Fax: +81-76-434-5010
E-mail: osmotani@ms.toyama-mpu.ac.jp

Title
The Presence of a Local Immune System in the Upper Blind and Lower Part of the Human Nasolacrimal Duct

Author
Paola SIRIGU, Cristina MAXIA, Roberto PUXEDDU, Ignazio ZUCCA, Franca PIRAS and Maria Teresa PERRA

Address
Department of Cytomorphology, Department of Surgical Sciences and Organ Transplantation-Section of Otolaryngology; and Clinic of Ophthalmology, University of Cagliari, Monserrato, Italy

Summary
The nasolacrimal duct is exposed to exogenous agents, including potentially harmful microorganisms, coming from the eye surface by the lacrimal sac, and from the nasal cavity by the inferior meatus of the nose. The upper blind and lower part of the human nasolacrimal duct were examined immunohistochemically to ascertain the presence and localization of immunoglobulin-producing cells and the epithelial expression of IgA, IgM, and IgG in order to verify the possible antimicrobial properties of this duct. IgA-, IgM-, and IgG-positive immunocompetent cells were recognizable in the lamina propria of the upper blind and lower part of the human nasolacrimal duct, while an evident immunoreactivity for sIgA, IgM, and IgG was demonstrated in the cytoplasm of the apical epithelial cells. The results suggest that all the effector components of the mucosal immune system are present in that area of the human nasal mucosa next to the opening of the nasolacrimal duct as well as in the human lacrimal sac.

Prof. Paola SIRIGU
Dipartimento di Citomorfologia Cittadella Universitaria
SS. 554-Bivio per Sestu
09042 Monserrato (CA)
Italy
Tel: +39 070-6754076
Fax: +39 070-6754003
E-mail: psirigu@unica.it

Title
Surface Remodeling Associated with Vasopressin-Induced Membrane Traffic in L6 Myogenic Cells

Author
Dario COLETTI, Simonetta PALLESCHI, Leopoldo SILVESTRONI, Francesco TOMEI, Mario MOLINARO and Sergio ADAMO

Address
Departments of Histology and Medical Embryology and of Medical Physiopathology, and Institute of Forensic Medicine, University "La Sapienza", Rome, Italy

Summary
The plasma membrane is dynamically remodeled as a function of the cell cycle, motility and membrane traffic. We have previously shown that arg8-vasopressin (AVP) stimulation of L6 myoblasts induces the activation of phosholipase D during the first minutes of stimulation, and the differentiation of 1,6 myoblasts as a long term effect. We now report that AVP also induces two types of morphological responses in L6 cells within a few minutes of stimulation: exocytosis, apparent as uncoated pits, and the generation of membrane projections and reffles. Thus, such an experimental model is suitable for the study of hormone-induced morphological surface modifications and their regulatory mechanisms. In L6 cells, AVP-induced projection generation depends on the integrity of microfilaments, intermediate filaments, and microtubules. Moreover, projection generation and exocytosis appear to be independently regulated phenomena: in fact, inhibition of the de novo synthesis of phosphatidylcholine inhibits membrane traffic but fails to block projection appearance. Conversely, the latter phenomenon, unlike exocytosis, is mediated by PI3-kinase signaling. Thus, AVP induces two early, independently regulated morphological modifications in L6 cells: exocytosis, involved in plasma membrane phospholipid turnover, and membrane projections, likely involved in cell migration.

Sergio ADAMO, M.D.
Department of Histology and
Medical Embryology
University "La Sapjenza"
via Scarpa 14
00161 Rome, Italy
Tel: +39-0649766756
Fax: +39-064462854
E-mail: adamo@uniromal.it

Title
The Ultrastructure of Skeletal and Smooth Muscle in Experimental Protein Malnutrition in Rats Fed a Low Protein Diet

Author
Masayo OUMI, Masayuki MIYOSHI and Torao YAMAMOTO

Address
Department of Anatomy and Nutrition Morphology, Graduate School of Health and Nutrition Sciences, Nakamura Gakuen University; and Department of Anatomy, School of Medicine, Fukuoka University, Fukuoka, Japan

Summary
Light microscopy of the pectoralis muscle of rats on a low protein diet did not show such morphological alterations as atrophy, degeneration, or sarcoplasmic edema, but electron microscopy occasionally demonstrated ultrastructural changes only in the sarcomeres of myofibrils. In the affected sarcomeres, the Z-line was disrupted and often showed a jagged structure. The Z-substance with electron opacity was frequently present flowing along the long axis of myofibrils, here referred to as the streaming of Z-lines. In addition, regular striations formed by the reciprocal arrangement of thick and thin filaments disappeared from the affected sarcomeres, though these filaments were still discernible. Two or more consecutive sarcomeres in a single myofibril were occasionally involved in these changes. A further two or more neighboring sarcomeres at the same level of myofibrils were affected transversely by these structural alterations. On the other hand, the ultrastructure of the intestinal smooth muscle was not affected by protein deficiency. The study suggests that the ultrastructural damage induced by a low protein diet is attributed to the activation of endogenous protease by the excess leaking of Ca2+ into the cytosol as a result of lipid peroxidation of cell membrane by raised free radicals, owing to the depletion of glutathione production by protein deficiency. It also suggests that the smooth muscle cells differ in their susceptibility to protein deficiency from the skeletal muscle cells.

Prof. Torao YAMAMOTO
Department of Anatomy and
Nutrition Morphology
Graduate School of Health and
Nutrition Sciences
Nakamura Gakuen University
5-7-1, Johnan-Ku, Fukuoka
814-0198 Japan
Tel: +81-92-851-2531
Fax: +81-92-841-7762
E-mail: yamamoto@cc.nakamura-u.ac.jp

Title
Enhanced Visualization of Weak Colloidal Iron Signals with Bodian's Protein Silver for Demonstration of Perineuronal Nets of Proteoglycans in the Central Nervous System

Author
Luo Jia HONG, Wafaa Alaa El-din MUBARAK, Yuko SUNAMI, Shinichiro MURAKAMI, Yasuhiro FUYAMA, Aiji OHTSUKA and Takuro MURAKAMI

Address
Departments of Anatomy and Otolaryngology, Faculty of Medicine, Okayama University Medical School, Okayama, Japan

Summary
The present study aimed for a clear visualization of faintly deposited colloidal iron in tissue sections for light microscopy. Paraffin blocks containing paraformaldehyde-fixed brain tissue from healthy adult mice were cut into sections 10-15 microm thick. After deparaffinization, the sections were stained with fine cationic iron colloid at a pH value of 1.0-1.5, and treated with a mixture of potassium ferrocyanide and hydrochloride for Prussian blue reaction. Some sections were further treated with Bodian's protein silver after the Prussian blue reaction. This sensitized development of Prussian blue reaction with Bodian's protein silver more clearly visualized the faintly deposited cationic colloidal irons than the demonstration by Prussian blue reaction alone, and allowed an enhanced visualization of the perineuronal nets of sulfated proteoglycans in the brain. Thus, such fine perineuronal sulfated proteoglycans as those in the CA3 field of the hippocampus, which are weakly stained with cationic iron colloid and usually overlooked by a demonstration with only a Prussian blue reaction, could be clearly visualized with striking contrast by the sensitized development with Bodian's protein silver after the Prussian blue reaction. Preliminary hyaluronidase digestion erased Bodian's protein silver development of perineuronal sulfated proteoglycans. Though some axonal fibers were also additionally stained with Bodian's protein silver itself, this sensitized development is useful to enhance such weak colloidal iron signals as are hardly detectable by only Prussian blue reaction.

Prof. Takuro MURAKAMI
Department of Anatomy
Faculty of Medicine
Okayama University Medical School
2-5-1 Shikata-cho, 0kayama
700-8558 Japan
Tel: +81-86-235-7088
Fax: +81-86-235-7095

Title
Morphology of the Glomerular Nerve Endings in the Dorsal Nasal Ligament of the Dog

Author
Yoshio YAMAMOTO, Yasuro ATOJI and Yoshitaka SUZUKI

Address
Laboratory of Veterinary Anatomy, Faculty of Agriculture, Gifu University, Gifu, Japan

Summary
The nasal atrium appears to be an important sensory site in the dog, yet no literature is available concerning its nerve supply. The present paper demonstrates the occurrence of glomerular nerve endings in the canine nasal atrium, using immunohistochemistry for neurofilament protein (NFP) and for glial fibrillary acidic protein (GFAP). Glomerular nerve endings occurred on the perichondrium of the septal and the dorsal lateral nasal cartilages, and their terminal portions were attached with dense collagen fibril strands of the dorsal nasal ligament. The glomerular endings were derived from a thick parent axon which branched repeatedly. Complicated winding nerve fibers gave rise to numerous thin filamentous terminals. Accumulations of GFAP immunoreactive glial cells were also observed. Immunoelectron microscopy for NFP revealed several axon terminals in the glomerular endings which contained numerous neurofilaments and mitochondria and were incompletely covered by Schwann cell sheaths. The glomerular endings in the dog nasal vestibule are suggested to perceive tensional changes in the nasal dorsal ligament caused by the opening of the nostrils and to be involved in the reflex regulating the activity of the nasal muscles.

Dr. Yoshio YAMAMOTO
Laboratory of Veterinary Anatomy
Department of Veterinary Science
Faculty of Agriculture, Gift; University
Yanagido 1-1, Gifu
501-1193 Japan
Tel: +81-58-293-2937
Fax: +81-58-293-2840
E-mail: yamamoto@cc.gifu-u.ac.jp

Title
Kupffer Cell Activation and Hematopoiesis in the Liver of Autoimmune MRL-lpr/lpr Mice

Author
Kenzo CHO, Shuichi SEKI, Kazuki NAKATANI, Kenzo KOBAYASHI and Kenji KANEDA

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

Summary
Hematopoiesis can be induced in the adult murine liver by the administration of macrophage activators. The proliferation of macrophages and extrathymic T cells is spontaneously induced in the liver of autoimmune MRL-lpr/lpr mice, and deeply involved in the development of disease. To study the role of Kupffer cell activation in the induction of hematopoiesis and lymphocyte proliferation in the liver, we histologically analysed the kinetic and spatial relationship between Kupffer cells and hematopoietic cells or lymphocytes. At 5 weeks of age before the onset of disease, there were no appreciable histological changes in the liver. At 7 weeks, Kupffer cells had slightly increased in number, while hematopoietic islands were not yet detected. When disease had fully developed at 14 weeks, Kupffer cells were considerably increased in number and size, and exhibited numerous lysosomes. Hematopoietic cells of erythroid and myeloid series frequently appeared in the sinusoid, and lay in close apposition to Kupffer cells. Promyelocytes further migrated into the space of Disse to cluster there, being surrounded by the stellate cells (or fat-storing cells) and hepatocytes. After maturation, metamyelocytes and mature granulocytes were released into the sinusoidal circulation. Mitotic figures were detected in the cells of both erythroid and myeloid series. Lymphocytes proliferated in various sites such as in the sinusoid lumen, the space of Disse, and interlobular connective tissue, whether associated or not with Kupffer cells. The present results indicate that erythropoiesis, granulopoiesis, and lymphocyte proliferation are induced in the liver of MRL-lpr/lpr mice and are closely associated with Kupffer cell activation.

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@msic.med.osaka-cu.ac.jp

Title
Intensely Negative-charged Pericapillary spaces in the Rat Pineal Gland

Author
Takehito TAGUCHI, Motoyasu KOSAKA, Shinichiro MURAKAMI, Aiji OHTSUKA and Takuro MURAKAMI

Address
Department of Radiological Technology, Faculty of Health Sciences; and Department of Anatomy, Faculty of Medicine, Okayama University Medical School, Okayama, Japan

Summary
Electron microscopy of ultrathin sections stained with cationic iron colloid revealed that the rat pineal gland is provided with wide and intensely negative-charged pericapillary spaces. Light microscopically, the negative charging of the pericapillary spaces was completely eliminated by digestion with hyaluronidase and chondroitinase ABC. This pericapillary negative charging was also erased by digestion with collagenase. The results indicate that the negative charging is derived from sulfated proteoglycans which are bound to collagen molecules. These sulfated proteoglycans in the pericapillary spaces may retain numerous water molecules to form a tissue gel, and so act as a selective sieve regulating the passage of tissue molecules.

Dr. Takehito TAGUCHI
Department of Radiological Technology
Faculty of Health Sciences
Okayama University Medical School
2-5-l Shikata-cho, Okayama
700-8558 Japan
Tel: +81-86-235-6903
Fax: +81-86-235-7095
E-mail: em2kai@med.okayama-u.ac.jp

Title
Effects of Short-term Denervation and Subsequent Reinnervation on Motor Endplates and the Soleus Muscle in the Rat

Author
Harutoshi SAKAKIMA, Seiichi KAWAMATA, Satoru KAI, Junya OZAWA and Natsue MATSVVRA

Address
Institute of Health Sciences, Faculty of Medicine, Hiroshima University, Hiroshima, Japan

Summary
The rat sciatic nerve was locally frozen, and changes in the nerve, motor endplates, and the soleus muscle were examined for up to 6 weeks by light and electron microscopy. The wet weights of denervated soleus muscles compared with contralateral values progressively declined to a minimum at 2 weeks after injury (60.7 +/- 2.5%) and began to reverse following 3 weeks. The sciatic nerve thoroughly degenerated after freezing. However, numerous regenerated myelinated and thin nerve fibers were observed at 3 weeks. They were considerably enlarged but still smaller than normal counterparts at 6 weeks postoperatively. Nerve terminals containing synaptic vesicles of endplates disappeared at day 1 and mostly reappeared at 3 weeks (about 70% of the endplates). All endplates examined were reinnervated at 4, 5, and 6 weeks. On the other hand, postsynaptic folds of muscle fibers seemed to be only slightly influenced by denervation or reinnervation. Ultrastructural alterations of myofibrils, in particular the loss of register, immediately appeared after denervation, spread progressively, peaked at 2 weeks, ameliorated following reinnervation, and became significantly normalized at 6 weeks after freezing. The proportion of type II fibers in the soleus muscle similary showed an increase and a decrease with a short delay in response to denervation and reinnervation, respectively. This study clearly demonstrated that the nerve supply affects the ultrastructural integrity of skeletal muscles. In addition, changes in the endplates and the soleus muscle evaluated in this study after short-term denervation are largely reversible following reinnervation.

Prof. Seiichi KAWAMATA
Basic Division of Physical Therapy
Institute of Health Sciences
Faculty of Medicine Hiroshima University
Kasumi 1-2-3, Hiroshima
734-8551 Japan
Tel: +81-82-257-5410
Fax: +81-82-257-5344
E-mail: kawamat@hiroshima-u.ac.jp