Arch. Histol. Cytol., Vol. 55, Suppl. (1992) p. 131-138
The
"Limiting
L. LUCIANO Laboratory
and
Ridge"
of the
Rat
Stomach*
E. REALE
of Cell Biology and Electron Microscopy, Hannover Medical School, Hannover, Federal Republic of Germany
Received February 5, 1992
Summary. from
In
the
the
glandular
rat,
the
forestomach
stomach
by
stomach mucosa which generates on the inner surface of the organ. deep
groove
which
is flanked
stomach and distally by microscopy and scanning that the stomach
the
a fold
is
separated
of
the
to the forestomach and to the "limiting ridge". Indeed the structure of this "ridge" has never been thoroughly investigated. In order to gain new information on this special region, the rat stomach was studied by light microscopy in semithin sections and by scanning electron microscopy (SEM) before and after parts of the groove and/or of the ridge were removed by mechanical microdissection.
fore-
the "limiting ridge" This ridge overlaps a
proximally
by
the
glandular stomach. electron microscopy
foreLight reveal
keratinized squamous epithelium of the foremerges into the columnar epithelium of the
glandular stomach at the bottom of the groove. Among the columnar cells of the distal wall of the groove are numerous brush cells. A remarkably thick lamina muscularis
mucosae
extends
liar architecture ence of numerous
of the brush
deep
into
the
ridge.
The
"limiting ridge" and the prescells in its distal wall suggest
that zone
the region not only between forestomach
represents the transitional and glandular stomach but
that
it might
specific
have
MATERIALS
a more
The stomachs of 6 rats (Wistar Hannover, male, 200250 g/b.w.) were used. The animals received food and water ad libitum before they were killed by decapitation. The organ was removed and halved. The plane of section in the glandular region ran 3-5 mm distally to the clearly visible "limiting ridge". The content of the stomach was removed and the internal surface was cleaned by several blasts of buffer (the same used for the fixative) from a jet bottle. The half of the stomach containing the f orestomach was then immersed in 3% glutaraldehyde diluted in 0.1 M Nacacodylate buffer, pH 7.2 and fixed for 2 h. Under a dissecting microscope the "limiting ridge" and the bordering mucosa appeared uniformly smooth over most of their extent. Only toward the esophagus orifice, the mucosa was corrugated and the "limiting ridge" was deeply inf olded. In some speci-
function.
The stomach of the rat is characterized by the presence of an extensive forestomach (ToEPFER, 1891). This region represents nearly a third of the organ; its internal surface is covered by a squamous epithelium which is separated from the columnar epithelium of the adjacent glandular region by a mucosal fold ("Grenzf alte": TOEPFER, 1891; "limiting ridge": ROBERT, 1971). This extends circumferentially from the large curvature to the lesser one, immediately beyond the esophagus, which joins the stomach at right angle (BOTHA, 1962). Toward the glandular stomach a deep groove underlies the ridge. It is at the bottom of this groove that the squamous epithelium of the f orestomach merges into the columnar epithelium (TOEPFER, 1891; GUIEYSSE-PELLISSIER, 1937; WATTEL and GEUZE, 1978). Although the rat stomach is currently used in morphological and gastroenterological studies (reviewed in ITo, 1987), little attention is generally paid *This work was supported
in part by the Deutsche
AND METHODS
pecu-
mens this inf olding was cut, which allowed the sample to be flattened; thus, its observation in SEM was facilitated. Small tissue blocks were severed from one stomach along the "limiting ridge". These were postfixed in a buffered 0504 solution and embedded in Epon for light microscopy. The semithin (1um-thick) sections were stained with alcalinized toluidine blue. All other specimens were processed for SEM according to the
Forschungsgemeinschaft 131
(SFB 280).
132
L. LUCIANo and E. REALE:
b
a
Fig. 1 a and b. Rat stomach: transition from the forestomach (FS) to the glandular stomach (GS). a. A fold of the mucosa (the "limiting ridge", LR) separates FS from GS. Between LR and GS is a deep groove (G). Arrows point to consistent layers of smooth muscle cells in continuity with the muscularis mucosae of FS and GS. b. The transition zone from the squamous keratinized epithelium of FS to the columnar epithelium of GS at the bottom of G. The arrowheads draw attention to the tongue-like projection formed by the keratinized layers of the epithelia covering proximal and distal slopes of the fold. MM smooth muscle cells of the lamina muscularis mucosae irradiating into the lamina propria of the fold. a: x50, b: x560
Limiting
OTOTO technique (KELLEYet al., 1973), which renders the specimen thoroughly conductive. After dehydration in a graded series of acetone, the specimens were critical-point dried in a CPD 020 device (Balzers, Liechtenstein) and mounted on metal stubs using conducting carbon paint (Leit-C, Neubauer, Munster, FRG). After primary examination in a Philips 505 SEM, some specimens were transferred to a dissecting microscope, and the groove overlapped by the "limiting ridge" was opened using tiny needles. The specimens were cleaned examined by SEM.
with an air jet and
re-
RESULTS Using light microscopy, the "limiting ridge" was identified as part of a fold of the mucosa covered by squamous keratinized epithelium. This was thick and indented by relatively deep papillae on the proximal slope of the fold; on the distal slope it was low and lying on a smooth lamina propria (Fig. la, b). The
Fig. 2. "Limiting ridge" (LR) separating the esophagus. x 14
Ridge
of Rat Stomach
133
keratinization was pronounced on the proximal surface of the fold, but gradually decreasing on the distal one until it ceased at the bottom of the groove. The two squamous epithelial layers of the fold slopes met above the opening of the groove, generating a remarkable tongue-like projection composed solely of keratinized cells (Fig. lb). The wall of the groove facing the distal slope of the mucosal fold consisted of one layer of columnar cells, which were low and with few (if any) apical secretory granules close to the bottom, but higher and with an increasing number of apical granules toward the stomach surface (Fig. 1b). A thick layer of smooth muscle cells lay in the scanty connective tissue of the lamina propria of the ridge (Fig. 1b). The layer below the epithelium of the proximal slope of the ridge was in continuity with the muscularis mucosae of the forestomach; the smooth muscle cells were mostly obliquely cut. The layer bordering the epithelium of the distal slope was in continuity with the muscularis mucosae of the glandular stomach; the cells were usually cut longitudi-
the forestomach
(FS) from the glandular
stomach
(GS). E opening of
134
L. LUCUANOand E. RIALE:
b
a
Fig. 3 a. U-shaped aspect of the "limiting ridge" (arrowheads) close to the esophageal orifice (E). b. Detail of Fig. 3a (asterisk) at higher magnification. Proximal slope of the "limiting ridge". The keratinized squamous layers of the epithelium are corrugated into a palisade-like pattern. a: x 13, b: x 53
nally (Fig. 1b). In addition, a further layer of smooth muscle cells covered the other two where they met below the ridge. Therefore, in cross sections, the ridge always had a thick muscularis mucosae. Before dissection, the limiting ridge appeared in SEM as a circumferentially extended structure with its maximum height close to the esophagus orifice, where it protruded toward the glandular stomach, assuming a U-form ("horse-shoe fashion", BOTHA, 1962) (Figs. 2, 3a). The ridge was lined with several layers of squamous keratinized cells superimposed like tiles according to the flow direction of the gastric contents, i.e., those located more proximally covered part of the underlying distal ones (Figs. 3b, 4, 5). The squamous epithelium of the ridge bordered a narrow slit (Fig. 4) corresponding to the point of contact or close approximation of the tongue-like keratinized projection of the ridge to the columnar epithelium of the glandular region. The squamous epithelium of the proximal slope of the ridge in places assumed a palisade-like pattern with low elevations running parallel to each other and orthogonal to the main orientation of the ridge,
and all showing about the same width (approximately 30um) (Figs. 3b, 5). As seen after microdissection, a similar palisadelike aspect was presented by the squamous epithelium covering the distal slope of the ridge (Fig. 6). The components of this palisade, however, were thinner (from about 6 to 10um) than those of the proximal slope, and additionally showed squamous cells superimposed from the bottom of the groove toward the surface (Fig. 6). Therefore, the keratinized part of the epithelium on the proximal slope had an orientation opposite that covering the distal slope. The two keratinized parts met along the free margin of the ridge and their confluence generated the tongue-like projection. The weaker keratinization of the squamous cells close to the groove bottom allowed a distinct identification of the single superimposed cells on the distal side of the ridge; the single cells were not distinct on the proximal side where keratinization was strong. When the limiting ridge was removed by microdissection, the epithelium of the distal wall of the groove
Fig. 6. Distal slope of the "limiting ridge" (LR). The apical part of the ridge has been broken away by microdissection. The epithelium shows a palisade-like pattern like that of the proximal slope (see Fig. 3b). The tile-like arrangement is directed from the bottom of the groove to its tip. Asterisk: material lying on the groove bottom (see Fig. lb), arrowheads: some brush cells on the distal wall of the groove. x 820
Limiting
Ridge of Rat Stomach
135
4
5
6 Fig. 4. The "limiting ridge" (LR) separating the forestomach (FS) from the glandular stomach (GS) hinders the view of the groove. The keratinized squamous cells of the tongue-like projection of the fold (see Fig. 1b) are arranged in several overlapping layers. Between these and the glandular region is a narrow slit (arrowheads). x 112 Fig. 5. Proximal cells. x 225 Fig. 6.
slope of the ridge.
Legend on the opposite page.
Tile-like
arrangement
(from
FS to GS) of the squamous
keratinized
136
L. LUCIANO and E. REALE:
was exposed along with a few gastric pit openings (Fig. 7). Two types of cells composed the epithelium on this surface of the groove. The first, and more numerous,
of irregular size and polygonal borders, bore a tight covering of microvilli (Fig. 8a, b). The second one usually had a remarkably smaller surface than the first, but distinctly longer and thicker microvilli (Fig.
b
8a Fig. 7. The "limiting ridge" has been removed by microdissection (asterisk, remnants of the ridge) exposing the distal surface of the groove. Among the columnar cells are numerous brush cells appearing as small protruberances (some marked by arrowheads) as well as the opening of a few gastric pits. x320 Fig. 8 a and b. Brush cells (arrowheads) scattered among the columnar cells at low (a) and high (b) magnifications. Both cell types bear microvilli which are longer and somewhat thicker on the brush cells than on the columnar cells. a: x2,300, b: x6,800
Limiting
8b). The first cell type were the columnar second the brush cells.
cells, the
DISCUSSION The results of the present investigation show that the "limiting ridge" not only represents the border be tween the forestomach and glandular stomach (TOEPFER, 1891; ROBERT, 1971; BIVIN et al., 1979; HEBEL and STROMBERG,1986), but probably has various and more complex functions, as suggested by its architecture and by the brush cells which are particularly numerous in this region. Concerning the architecture, the epithelium of the ridge toward the forestomach is extensively keratinized and deeply indented with the lamina propria. Both these aspects gradually become less evident on the distal slope of the ridge and finally disappear close to the bottom of the groove. This suggests that the distal slope of the ridge is less exposed than the proximal one the severe mechanical stimuli, such as those generated by the food intake of the animal. A further morphological characteristic is the lamina muscularis mucosae, which extends very close to the epithelium and, since it is formed by the confluence of the laminae of the forestomach and of the glandular stomach, is also unusually thick. The different orientation of the smooth muscle cell layers suggests that various effects can be brought about through their contraction. For example, access to the adjacent groove can be varied; stiffening of the ridge itself can be induced, which causes the palisade-like corrugation of the epithelium and thus hinders the irregular collapse of the ridge and groove. The "limiting ridge" becomes more prominent near the esophagus. It has been shown that through shifting the two sides of the U-shaped inf olding, the esophagus orifice opens or closes, suggesting an intrinsic force in the fold and that the thick muscularis mucosae is the only structure responsible "to maintain independent tone in the fold that guards the cardia orifice" (BOTHA, 1958). Conceivably, the contraction of the muscularis mucosae extends along the whole "limiting ridge", causing various degrees of circumferential stretch in this transition zone of the stomach. Numerous brush cells have been described in the distal wall of the groove using both light and electron microscopy (ISOMAKI, 1973; WATTEL and GEUZE, 1978; ISEKI and KONDO, 1989). The function of these cells is controversial. One hypothesis-based 1) on their synaptic contact with afferent terminals in the developing trachea of the rat (LUCIANo et al., 1968)
Ridge
of Rat
Stomach
137
and 2) on their occurrence in several epithelia undergoing the influence of mechanical forces (LUCIANo and REALE, 1990)-suggests that they are sensory cells. The peculiar location of a large number of brush cells in a groove covered by a mucosal fold, as observed in the present study, indicates that these cells may be shielded or protected from severe mechanical stimuli. In addition, the slit along the "limiting ridge" could be closed or opened, modulating the diffusion of the glandular stomach contents into the groove. Therefore, the stimulation of the brush cells could be both variable and gradual. In the rat, unlike in man and carnivores, but similar to several other monogastric mammals (the pig, horse, and a large number of rodents), a forestomach of varying extent has been described (TOEPFER,1891; ELLENBERGERand v. SCHUMACHER, 1914; BOTHA, 1962). However, this part of the stomach (in the rat, nearly 1/3) has been mostly disregarded since it is considered simply a reservoir (BIvIN et al., 1979). However, it is well known that in non monogastric mammals, the rumen, for instance, besides having the function of a reservoir also fulfills other tasks; its squamous epithelium is able to absorb ions by an active mechanism (MARTENS et al., 1991); in its interior, essential microbial digestive processes and the sorting of solid food particles from the fluid continuously occur (v. ENGELHARDTet al., 1986). Therefore, as already suggested a century ago by TOEPFER (1891), the f orestomach in rodents could be the site where "eine vorlaufige Maceration findet statt" (a preliminary maceration occurs). In addition, the "limiting ridge" could be related to the movement of fluid contents between the f orestomach stomach.
and glandular
Acknowledgements. We would like to thank Ms S. FRYK for correcting the English manuscript.
REFERENCES
BIVIN, W. S., M. P. CRAWFORD and N. R. BREWER: Morphophysiology. In: (ed. by) H. J. BAKER,J. R. LINDSEY and S. H. WEISBROTH: The laboratory rat, Vol. I. Academic Press, New York-London-Toronto-SydneySan Francisco, 1979 (p. 74-104). BOTHA, G. S. M.: Mucosal folds at the cardia as a component of the gastro-oesophageal closing mechanism. Brit. J. Surg. 45: 569-580 (1958). -: The gastro-oesophageal junction. Little, Brown and Company, Boston, 1962. ELLENBERGER, W. and S. v. SCHUMACHER: Histologie der Haussaugetiere, 4. Aufl. Paul Parey, Berlin, 1914.
138 L. LUCIAANO and E. REALE
ENGELHARDT, W. v., M. LECHNER-DOLL, R. HELLER, H. J. SCHWARTZ, T. RUTAGWENDA and W. SCHULTKA: Physiology of the forestomach in camelids with particular reference to adaptation to extreme dietary conditions. A comparative approach. Zool. Beitr. N. F. (Suppl.) 30: 1-15 (1986). GUIEYSSE-PELLISSIER, A.: Note sur la keratinisation de 1'estomac du rat et de la souris. C. R. Assoc. Anat. 32: 206-211 (1937). HEBEL, R. and M. W. STROMBERG:Anatomy and embryology of the laboratory rat. BioMed, Worthsee, 1986. ISEKI, S. and H. KONDO: Specific localization of hepatic fatty acid-binding protein in the gastric brush cells of rats. Cell Tiss. Res. 257: 545-548 (1989). SOMAKI, A. M.: A new cell type (tuft cell) in the gastrointestinal mucosa of the rat. Acta Pathol. Microbiol. Scand. Sect. A (Suppl.) 240: 1-35 (1973). ITO, S.: Functional gastric morphology. In: (ed. by) L. R. JOHNSON: Physiology of the gastrointestinal tract, Vol. 1, 2nd. ed., Raven Press, New York, 1987 (p. 817851). KELLEY, R. o., R. A. DEKKER and J. G. BLUEMINK: Ligand-mediated osmium binding: Its application in coating biological specimens for scanning electron microscopy. J. Ultrastr. Res. 45: 254-258 (1973). LUCIANO, L. and E. REALE: Brush cells of the mouse gallbladder. A correlative light and electron-microscopical study. Cell Tiss. Res. 262: 339-349 (1990).
LUCIANO, L., E. "chemorezeptive"
REALE and H. RUSKA: Uber eine Sinneszelle in der Trachea der Ratte.
Z. Zellforsch. 85: 350-375 (1968). MARTENS, H., G. LABEL and B. STROZYK: Mechanism of electrically silent Na and Cl transport across the rumen epithelium of sheep. Exp. Physiol. 76: 103-114 (1991). ROBERT, A.: Proposed terminology for the anatomy of the rat stomach. Gastroenterology 60: 344-345 (1971). TOEPFER, K.: Die Morphologie des Magens der Rodentia. Morphol. Jahrb. 17: 380-407 (1891. WATTEL, W. and J. J. GEUZE: The cells of the rat gastric groove and cardia. An ultrastructural and carbohydrate histochemical study, with special reference to the fibrillovesicular cells. Cell Tiss. Res. 186: 375-391 (1978).
Prof.
Dr. Liliana
LucIANo
Abteilung Zellbiologie and Elektronenmikroskopie Medizinische Hochschule Hannover Konstanty-Gutschow-Str.
8
3000 Hannover 61 Federal Republic of Germany
The
"Limiting
L. LUCIANO Laboratory
and
Ridge"
of the
Rat
Stomach*
E. REALE
of Cell Biology and Electron Microscopy, Hannover Medical School, Hannover, Federal Republic of Germany
Received February 5, 1992
Summary. from
In
the
the
glandular
rat,
the
forestomach
stomach
by
stomach mucosa which generates on the inner surface of the organ. deep
groove
which
is flanked
stomach and distally by microscopy and scanning that the stomach
the
a fold
is
separated
of
the
to the forestomach and to the "limiting ridge". Indeed the structure of this "ridge" has never been thoroughly investigated. In order to gain new information on this special region, the rat stomach was studied by light microscopy in semithin sections and by scanning electron microscopy (SEM) before and after parts of the groove and/or of the ridge were removed by mechanical microdissection.
fore-
the "limiting ridge" This ridge overlaps a
proximally
by
the
glandular stomach. electron microscopy
foreLight reveal
keratinized squamous epithelium of the foremerges into the columnar epithelium of the
glandular stomach at the bottom of the groove. Among the columnar cells of the distal wall of the groove are numerous brush cells. A remarkably thick lamina muscularis
mucosae
extends
liar architecture ence of numerous
of the brush
deep
into
the
ridge.
The
"limiting ridge" and the prescells in its distal wall suggest
that zone
the region not only between forestomach
represents the transitional and glandular stomach but
that
it might
specific
have
MATERIALS
a more
The stomachs of 6 rats (Wistar Hannover, male, 200250 g/b.w.) were used. The animals received food and water ad libitum before they were killed by decapitation. The organ was removed and halved. The plane of section in the glandular region ran 3-5 mm distally to the clearly visible "limiting ridge". The content of the stomach was removed and the internal surface was cleaned by several blasts of buffer (the same used for the fixative) from a jet bottle. The half of the stomach containing the f orestomach was then immersed in 3% glutaraldehyde diluted in 0.1 M Nacacodylate buffer, pH 7.2 and fixed for 2 h. Under a dissecting microscope the "limiting ridge" and the bordering mucosa appeared uniformly smooth over most of their extent. Only toward the esophagus orifice, the mucosa was corrugated and the "limiting ridge" was deeply inf olded. In some speci-
function.
The stomach of the rat is characterized by the presence of an extensive forestomach (ToEPFER, 1891). This region represents nearly a third of the organ; its internal surface is covered by a squamous epithelium which is separated from the columnar epithelium of the adjacent glandular region by a mucosal fold ("Grenzf alte": TOEPFER, 1891; "limiting ridge": ROBERT, 1971). This extends circumferentially from the large curvature to the lesser one, immediately beyond the esophagus, which joins the stomach at right angle (BOTHA, 1962). Toward the glandular stomach a deep groove underlies the ridge. It is at the bottom of this groove that the squamous epithelium of the f orestomach merges into the columnar epithelium (TOEPFER, 1891; GUIEYSSE-PELLISSIER, 1937; WATTEL and GEUZE, 1978). Although the rat stomach is currently used in morphological and gastroenterological studies (reviewed in ITo, 1987), little attention is generally paid *This work was supported
in part by the Deutsche
AND METHODS
pecu-
mens this inf olding was cut, which allowed the sample to be flattened; thus, its observation in SEM was facilitated. Small tissue blocks were severed from one stomach along the "limiting ridge". These were postfixed in a buffered 0504 solution and embedded in Epon for light microscopy. The semithin (1um-thick) sections were stained with alcalinized toluidine blue. All other specimens were processed for SEM according to the
Forschungsgemeinschaft 131
(SFB 280).
132
L. LUCIANo and E. REALE:
b
a
Fig. 1 a and b. Rat stomach: transition from the forestomach (FS) to the glandular stomach (GS). a. A fold of the mucosa (the "limiting ridge", LR) separates FS from GS. Between LR and GS is a deep groove (G). Arrows point to consistent layers of smooth muscle cells in continuity with the muscularis mucosae of FS and GS. b. The transition zone from the squamous keratinized epithelium of FS to the columnar epithelium of GS at the bottom of G. The arrowheads draw attention to the tongue-like projection formed by the keratinized layers of the epithelia covering proximal and distal slopes of the fold. MM smooth muscle cells of the lamina muscularis mucosae irradiating into the lamina propria of the fold. a: x50, b: x560
Limiting
OTOTO technique (KELLEYet al., 1973), which renders the specimen thoroughly conductive. After dehydration in a graded series of acetone, the specimens were critical-point dried in a CPD 020 device (Balzers, Liechtenstein) and mounted on metal stubs using conducting carbon paint (Leit-C, Neubauer, Munster, FRG). After primary examination in a Philips 505 SEM, some specimens were transferred to a dissecting microscope, and the groove overlapped by the "limiting ridge" was opened using tiny needles. The specimens were cleaned examined by SEM.
with an air jet and
re-
RESULTS Using light microscopy, the "limiting ridge" was identified as part of a fold of the mucosa covered by squamous keratinized epithelium. This was thick and indented by relatively deep papillae on the proximal slope of the fold; on the distal slope it was low and lying on a smooth lamina propria (Fig. la, b). The
Fig. 2. "Limiting ridge" (LR) separating the esophagus. x 14
Ridge
of Rat Stomach
133
keratinization was pronounced on the proximal surface of the fold, but gradually decreasing on the distal one until it ceased at the bottom of the groove. The two squamous epithelial layers of the fold slopes met above the opening of the groove, generating a remarkable tongue-like projection composed solely of keratinized cells (Fig. lb). The wall of the groove facing the distal slope of the mucosal fold consisted of one layer of columnar cells, which were low and with few (if any) apical secretory granules close to the bottom, but higher and with an increasing number of apical granules toward the stomach surface (Fig. 1b). A thick layer of smooth muscle cells lay in the scanty connective tissue of the lamina propria of the ridge (Fig. 1b). The layer below the epithelium of the proximal slope of the ridge was in continuity with the muscularis mucosae of the forestomach; the smooth muscle cells were mostly obliquely cut. The layer bordering the epithelium of the distal slope was in continuity with the muscularis mucosae of the glandular stomach; the cells were usually cut longitudi-
the forestomach
(FS) from the glandular
stomach
(GS). E opening of
134
L. LUCUANOand E. RIALE:
b
a
Fig. 3 a. U-shaped aspect of the "limiting ridge" (arrowheads) close to the esophageal orifice (E). b. Detail of Fig. 3a (asterisk) at higher magnification. Proximal slope of the "limiting ridge". The keratinized squamous layers of the epithelium are corrugated into a palisade-like pattern. a: x 13, b: x 53
nally (Fig. 1b). In addition, a further layer of smooth muscle cells covered the other two where they met below the ridge. Therefore, in cross sections, the ridge always had a thick muscularis mucosae. Before dissection, the limiting ridge appeared in SEM as a circumferentially extended structure with its maximum height close to the esophagus orifice, where it protruded toward the glandular stomach, assuming a U-form ("horse-shoe fashion", BOTHA, 1962) (Figs. 2, 3a). The ridge was lined with several layers of squamous keratinized cells superimposed like tiles according to the flow direction of the gastric contents, i.e., those located more proximally covered part of the underlying distal ones (Figs. 3b, 4, 5). The squamous epithelium of the ridge bordered a narrow slit (Fig. 4) corresponding to the point of contact or close approximation of the tongue-like keratinized projection of the ridge to the columnar epithelium of the glandular region. The squamous epithelium of the proximal slope of the ridge in places assumed a palisade-like pattern with low elevations running parallel to each other and orthogonal to the main orientation of the ridge,
and all showing about the same width (approximately 30um) (Figs. 3b, 5). As seen after microdissection, a similar palisadelike aspect was presented by the squamous epithelium covering the distal slope of the ridge (Fig. 6). The components of this palisade, however, were thinner (from about 6 to 10um) than those of the proximal slope, and additionally showed squamous cells superimposed from the bottom of the groove toward the surface (Fig. 6). Therefore, the keratinized part of the epithelium on the proximal slope had an orientation opposite that covering the distal slope. The two keratinized parts met along the free margin of the ridge and their confluence generated the tongue-like projection. The weaker keratinization of the squamous cells close to the groove bottom allowed a distinct identification of the single superimposed cells on the distal side of the ridge; the single cells were not distinct on the proximal side where keratinization was strong. When the limiting ridge was removed by microdissection, the epithelium of the distal wall of the groove
Fig. 6. Distal slope of the "limiting ridge" (LR). The apical part of the ridge has been broken away by microdissection. The epithelium shows a palisade-like pattern like that of the proximal slope (see Fig. 3b). The tile-like arrangement is directed from the bottom of the groove to its tip. Asterisk: material lying on the groove bottom (see Fig. lb), arrowheads: some brush cells on the distal wall of the groove. x 820
Limiting
Ridge of Rat Stomach
135
4
5
6 Fig. 4. The "limiting ridge" (LR) separating the forestomach (FS) from the glandular stomach (GS) hinders the view of the groove. The keratinized squamous cells of the tongue-like projection of the fold (see Fig. 1b) are arranged in several overlapping layers. Between these and the glandular region is a narrow slit (arrowheads). x 112 Fig. 5. Proximal cells. x 225 Fig. 6.
slope of the ridge.
Legend on the opposite page.
Tile-like
arrangement
(from
FS to GS) of the squamous
keratinized
136
L. LUCIANO and E. REALE:
was exposed along with a few gastric pit openings (Fig. 7). Two types of cells composed the epithelium on this surface of the groove. The first, and more numerous,
of irregular size and polygonal borders, bore a tight covering of microvilli (Fig. 8a, b). The second one usually had a remarkably smaller surface than the first, but distinctly longer and thicker microvilli (Fig.
b
8a Fig. 7. The "limiting ridge" has been removed by microdissection (asterisk, remnants of the ridge) exposing the distal surface of the groove. Among the columnar cells are numerous brush cells appearing as small protruberances (some marked by arrowheads) as well as the opening of a few gastric pits. x320 Fig. 8 a and b. Brush cells (arrowheads) scattered among the columnar cells at low (a) and high (b) magnifications. Both cell types bear microvilli which are longer and somewhat thicker on the brush cells than on the columnar cells. a: x2,300, b: x6,800
Limiting
8b). The first cell type were the columnar second the brush cells.
cells, the
DISCUSSION The results of the present investigation show that the "limiting ridge" not only represents the border be tween the forestomach and glandular stomach (TOEPFER, 1891; ROBERT, 1971; BIVIN et al., 1979; HEBEL and STROMBERG,1986), but probably has various and more complex functions, as suggested by its architecture and by the brush cells which are particularly numerous in this region. Concerning the architecture, the epithelium of the ridge toward the forestomach is extensively keratinized and deeply indented with the lamina propria. Both these aspects gradually become less evident on the distal slope of the ridge and finally disappear close to the bottom of the groove. This suggests that the distal slope of the ridge is less exposed than the proximal one the severe mechanical stimuli, such as those generated by the food intake of the animal. A further morphological characteristic is the lamina muscularis mucosae, which extends very close to the epithelium and, since it is formed by the confluence of the laminae of the forestomach and of the glandular stomach, is also unusually thick. The different orientation of the smooth muscle cell layers suggests that various effects can be brought about through their contraction. For example, access to the adjacent groove can be varied; stiffening of the ridge itself can be induced, which causes the palisade-like corrugation of the epithelium and thus hinders the irregular collapse of the ridge and groove. The "limiting ridge" becomes more prominent near the esophagus. It has been shown that through shifting the two sides of the U-shaped inf olding, the esophagus orifice opens or closes, suggesting an intrinsic force in the fold and that the thick muscularis mucosae is the only structure responsible "to maintain independent tone in the fold that guards the cardia orifice" (BOTHA, 1958). Conceivably, the contraction of the muscularis mucosae extends along the whole "limiting ridge", causing various degrees of circumferential stretch in this transition zone of the stomach. Numerous brush cells have been described in the distal wall of the groove using both light and electron microscopy (ISOMAKI, 1973; WATTEL and GEUZE, 1978; ISEKI and KONDO, 1989). The function of these cells is controversial. One hypothesis-based 1) on their synaptic contact with afferent terminals in the developing trachea of the rat (LUCIANo et al., 1968)
Ridge
of Rat
Stomach
137
and 2) on their occurrence in several epithelia undergoing the influence of mechanical forces (LUCIANo and REALE, 1990)-suggests that they are sensory cells. The peculiar location of a large number of brush cells in a groove covered by a mucosal fold, as observed in the present study, indicates that these cells may be shielded or protected from severe mechanical stimuli. In addition, the slit along the "limiting ridge" could be closed or opened, modulating the diffusion of the glandular stomach contents into the groove. Therefore, the stimulation of the brush cells could be both variable and gradual. In the rat, unlike in man and carnivores, but similar to several other monogastric mammals (the pig, horse, and a large number of rodents), a forestomach of varying extent has been described (TOEPFER,1891; ELLENBERGERand v. SCHUMACHER, 1914; BOTHA, 1962). However, this part of the stomach (in the rat, nearly 1/3) has been mostly disregarded since it is considered simply a reservoir (BIvIN et al., 1979). However, it is well known that in non monogastric mammals, the rumen, for instance, besides having the function of a reservoir also fulfills other tasks; its squamous epithelium is able to absorb ions by an active mechanism (MARTENS et al., 1991); in its interior, essential microbial digestive processes and the sorting of solid food particles from the fluid continuously occur (v. ENGELHARDTet al., 1986). Therefore, as already suggested a century ago by TOEPFER (1891), the f orestomach in rodents could be the site where "eine vorlaufige Maceration findet statt" (a preliminary maceration occurs). In addition, the "limiting ridge" could be related to the movement of fluid contents between the f orestomach stomach.
and glandular
Acknowledgements. We would like to thank Ms S. FRYK for correcting the English manuscript.
REFERENCES
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138 L. LUCIAANO and E. REALE
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Prof.
Dr. Liliana
LucIANo
Abteilung Zellbiologie and Elektronenmikroskopie Medizinische Hochschule Hannover Konstanty-Gutschow-Str.
8
3000 Hannover 61 Federal Republic of Germany
