Yonago Acta medica 2000;43:19–25

Scanning Electron Microscopic Observations on the IntracellularStructures of the Ciliated Tracheal Epithelium—Especially on theMorphological Differences between Conventional Rats and SpecificPathogen-Free Rats
Miwako Hanamoto and Takao Inoué
Second Department of Anatomy, Faculty of Medicine, Tottori University, Yonago 683-0826,
Japan
The three-dimensional architecture of the intracellular structures of the tracheal ciliatedepithelium was studied by scanning electron microscopy, paying particular attention
to the morphological difference between conventional rats and specific pathogen-free(SPF) rats. The surface of the trachea was densely covered with cilia in conventionalrats, but less in SPF rats. The smooth endoplasmic reticulum (ER) formed a threedimensional tubular meshwork under the basal bodies in both rats. In conventionalrats, the Golgi apparatus was highly developed and many Golgi vesicles were attachedto the lateral margin of the Golgi cisternae. In addition, the rough ER spread aroundthe nucleus. In the SPF rats, however, the Golgi appatatus was not so highly developed
and the rough ER was scarcely visible. The development of the Golgi apparatus andrough ER observed in the conventional rats indicate the active protein synthesis for theformation of the cilia which plays an important role in antimicrobial defense mechanisms.
Key words: ciliated epithelium; intracellular structure; scanning electron microscopy; specificpathogen-free rat; trachea

zymatic activity such as acethylcholinesterase(Graf and Stockinger, 1966), glucose-6-phosphatase (Kanamura, 1975; Hume and Burchell,1996) and peroxidase (Kataoka, 1971; Watanabe,1980; Christensen et al., 1981; Aoki et al., 1986;
Sakai et al., 1989; Kinbara et al., 1992). In
particular, acetylcholinesterase in the ER beneath the basal body is closely associated withthe ciliary movement. Additionally, the endogenous peroxidase activity has not been localizedin tracheal mucosal epithelial cells of specificpathogen-free (SPF) rats, but it became localized in the epithelial cells after natural infection
with Micoplasma pulmonis (Kinbara et al.,1992).
In this study, we studied the three-dimensional architecture of the intracellular structures

Although laboratory animals are frequently
exposed to a variety of microorganisms, little
attention has been paid in morphologicalstudies to the conditions in which the animals
were kept. The surface of the trachea is densely
covered with ciliated cells which play an important role in eliminating foreign bodies by ciliary
movement, when rats are kept under standard
laboratory conditions (Alexander et al., 1975).
In contrast, less ciliated cells are observed whenrats are housed in germ free conditions (Jefferyand Reid, 1975). It is then easily speculated
that the ultrastructures of ciliated cells are different between animals raised in conventional
conditions and pathogen-free ones.
The smooth endoplasmic reticulum (ER),distributed all over the ciliated cells, shows en-Abbreviations: DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; SEM, scanning electron microscopy; SPF, specific pathogen-free; TEM, transmission electron microscopy

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M. Hanamoto and T. InouéMM*a

10 µmb*
10 µmFig. 1. Longitudinally fractured surfaces of the tracheal epithelial cells of a conventional rat (a) and a
specific-pathogen free (SPF) rat (b). a: The surface of the epithelium is uniformly covered with cilia.
Mucous cells (M) are often visible between the ciliated cells. × 2,400. b: Ciliated cells and non-ciliated cells
are visible. The apical surface of the non-ciliated cells are covered with short microvilli. Mucous cells are
sometimes seen. Asterisks show the perinuclear intracellular structures exposed by the removal of thenucleus during the specimen preparation procedure. × 2,800.

by scanning electron microscopy (SEM), paying particular attention to the morphologicaldifferences between conventional rats and SPF
ones.

spp., Spironucleus spp., Trichomonas spp. andEntamoeba spp.
SEM specimens were fundamentally prepared by the A-O-D-O method (Tanaka andMitsushima, 1984). After the animals wereanaesthetized with an intraperitoneal injectionof Nembutal (50 mg/kg body weight), they
were fixed by perfusion with a mixture of 0.5%
glutaraldehyde and 0.5% formaldehyde (1/10mol/L phosphate buffer, pH 7.4). The tracheas
were then removed from the animals and excised into small pieces. After rinsing with thebuffer, they were immersed in 25% and 50%
dimethyl sulfoxide (DMSO) for 30 min each.
The specimens were frozen on a metal platechilled with liquid nitrogen and fractured into
two pieces using a razor blade and a hammer.
The fractured pieces were then placed in 50%
DMSO solution and thawed at room temperature. After they were completely rinsed in thebuffer, they were placed in 0.1% osmium tetroxide solution (1/10 mol/L phosphate buffer,Materials and methods
Ten female adult Wistar rats conventionallyraised on standard laboratory chow and water
ad libitum were used as the control animals.
Ten female adult Wistar SPF rats, purchasedfrom Japan SLC, Ind. (Shizuoka, Japan), wereused as the experimental animals. The SPF rats
were selected from a carefully monitored stock
declared free of the following specific pathogens: Sendai virus, Sialodacryoadenitis virus,
Hanta virus, Psedomonas aeruginosa, Salmonella
spp., Pasteurella pneumotropica, Bordetella
bronchiseptica, Streptococcus pneumoniae,
Corynebacterium kutscheri, Mycoplasma spp.,
Tyzzer’s organism, Syphacia spp., Giardia
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SEM of tracheal ciliated cellCytoplasmic matrices which prohibit thevisualization of intracellular structures werecompletely removed by the osmic maceration
procedure of the A-O-D-O method; consequently, both the outer and intracellularstructures of ciliated cells could be demonstrated in three dimensions by SEM.

pH 7.4) and left standing for 3 days or more at
20˚C. After rinsing with the buffer, the specimens were postfixed in 1% osmium tetroxidefor 1 to 2 h. They were then rinsed and treatedwith 1% tannic acid and 1% osmium tetroxide
for conductive staining (Murakami, 1974). Thespecimens were dehydrated through a gradedethanol series, replaced with t-butyl alcohol andfinally dried in a freeze-drier (ES-2020, HitachiLtd., Tokyo, Japan) (Inoué and Osatake, 1988).
The dried specimens were coated with platinumof about 2 nm by an ion coater with a rotatingstage (VX-10R, EIKO Engineering Co. Ltd.,
Mito, Japan) and observed with a field emission
SEM (S-4500, Hitachi Ltd.) operated at 7 to 15
kV.

Common ultrastructural findings of bothconventional and SPF rats
Basal bodies, extensions of the interior of thecilia, were lined beneath the apical plasma
membrane (Fig. 2). The smooth ER formed athree-dimensional tubular meshwork under thebasal bodies comprised of tubules of about 70 to80 nm in diameter. The nucleus, located in thebasal portion of the ciliated cells, was sometimes removed during the specimen preparation
procedure, thus the perinuclear structures could
be often observed. The Golgi apparatus, which
was composed of vesicles, vacuoles andparallel-arranged cisternae, was located in theResults
The surface of the trachea was covered withthree types of epithelial cells: ciliated, nonciliated and mucous cells (Fig. 1).

a1 µmb

1 µmFig. 2. Fractured surfaces of the apical portion of tracheal ciliated cells of a conventional rat (a) and SPF rat(b). Basal bodies are lined beneath the apical cell membrane. The tubular smooth endoplasmic reticulum
forms a three-dimensional meshwork, partly trapping the mitochondria. Microvilli are more numerous in theSPF rat than in the conventional rat. a: × 36,000. b: × 26,000.

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