A Scanning Electron Microscopic Study of Normal Human Oxyntic Mucosa Using Blunt Dissection and Freeze Fracture P E T E R A. M A C K E R C H E R , MD, K E V I N J. I V E Y , MD, F A C P , W I L L I A M N. B A S K I N , MD, and W I L L I A M J. K R A U S E , PhD
Biopsies from the fundic mucosa of healthy volunteers were examined by scanning electron microscopy following blunt dissection and freeze fracture. The mucosal smface exhibited a cobblestone appearance. With increased magnification, microvilli could be demonstrated on the luminal surface of individual surface cells. Blunt dissection of the gastric mucosa revealed tubular gastric pits descending from the surface until they opened into branched tubular gastric glands. The gastric glands are irregular in outline due to the knoblike projections of individual parietal cells. The internal structure of the component cells also was examined with the scanning electron microscope following freeze fracture. Mucous granules were observed in the apical portion of surface epithelial cells. Lumina of parietal cell canaliculi were found to be continuous with the lumen of the gastric glands. Pepsinogen granules could be seen throughout the cytoplasm of chief cells. The blunt dissection-freeze fracture technique utilizing the scanning electron microscope allows, for the first time, a three-dimensional view of human gastric mucosa, including the gastric pits and glands as well as some of the internal architecture of component cells.
C h a n g e s in g a s t r i c m u c o s a l u l t r a s t r u c t u r e h a v e been increasingly used to study pathological mucosa and the harmful effects of drugs on normal mucosa (1-8). Light microscopy (LM) and transmission electron m i c r o s c o p y (TEM) have been the m a i n s t a y o f s u c h s t u d i e s in b o t h a n i m a l s and man (1-5). More recently, scanning electron mic r o s c o p y (SEM) has added a new dimension to the demonstration of changes in surface epithelium (68). N e w techniques have now been developed to allow the examination of subepithelial structures and
cellular components with the SEM, by fracturing t h e t i s s u e or b y p r e p a r i n g it with b l u n t dissection (9-12). We have used these techniques to examine directly the three-dimensional structure of the human gastric mucosa. Previous structural concepts have been derived from reconstruction of serial sections studied by light and transmission electron microscopy.
From the Departments of Medicine and Anatomy, Hany S. Truman Memorial Veterans Hospital, and University of Missouri, Columbia, Missouri. This work was supported in part by the Medical Research Service of the Veterans Administration and grants from Smith, Kline and French Laboratories, as well as the patient study facilities of the UMMC Clinical Research Center. The technical assistance of Mr. G. E. Jeffrey is gratefully acknowledged. Address for reprint requests: Dr. K. J. Ivey, Division of Gastroenterology, Department of Medicine, University of Missouri Medical Center, Columbia, Missouri 65201.
Healthy male volunteers, aged 20-24 years, were studied after obtaining informed consent and institutional approval from the Human Experimentation Committee. After a 12-hr overnight fast, a nasogastric tube was passed and gastric contents aspirated. The stomach was then lavaged with 100 cc of 150 mM NaC1. A Quinton hydraulic multibiopsy tube was passed orally and positioned fluoroscopically in the gastric fundus. Biopsies, measuring 2 mm x 2 mm, were flushed from the stomach using tyrode solution under COz pressure. Duplicate biopsies were
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Digestive Diseases, Vol. 23, No. 5 (May 1978) 0002-9211/78/0500-0449505.00/1 9 1978 Digestive Disease Systems, Inc.
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Fig 1. (A) Light microscopy section of fundic mucosa. The apices of the cells are filled with dark staining mucous granules, and have a convex surface corresponding to "cobblestone effect" of SEM in Figure lB. (x500). (B) Scanning electron microscopy of gastric mucosa. Gastric pits (P) are seen and the surface epithelial cells give a cobblestone appearance. (z500).
taken from the same tube location, within seconds of each other (8). Tissues for SEM were fixed for 4 hr at 0~ C in 3.5% gluteraldehyde buffered in 0.1 M phosphate to pH 7.4. After washing in buffer, they were postfixed in 1% osmium tetroxide at 0 ~ C for 2 hr. The tissues were then blunt-dissected using a scalpel blade to cut one edge of the biopsy. The sides of the incision were then grasped with forceps and the biopsy carefully torn apart to demonstrate the gastric pits and gastric glands prior to critical-point drying, in a fashion similar to that of Hattori (9, 10) and Miller and Revel (11). The tissues were then dehydrated in alcohol and transferred to amyl acetate prior to critical point drying in liquid CO2. Additional tissues after similar fixation procedures were transferred through graded concentrations of ethanol to 100% ethanol, then dropped in a small container filled with liquid nitrogen to fractl~re the specimen for observation of intracellular structures (12). The fractured specimens were critical-point dried by the same method as the blunt-dissected specimens. The dried tissue was then placed on spinner stubs and coated with gold palladium alloy to a depth of 200 A in a vacuum evaporator and viewed with a Cambridge Stereoscope Mark II electron microscope.
450
Duplicate tissue samples were initially fixed for light microscopy and transmission electron microscopy, as were the specimens for scanning electron microscopy. After fixing or initial preparation, tissues were washed twice in 0.1 M phosphate buffer and processed for embedding in Epon 812. Thick sections (1-3/zm) of this material cut for light microscopy were stained in a 1 : 1 solution of 1% toluidine blue and 1% sodium borate. The sections were cut along the greatest length of the specimen. Ultrathin sections of the Epon embedded material cut for elect r o n m i c r o s c o p y w e r e m o u n t e d on u n c o a t e d g r i d s , stained with uranyl acetate and lead citrate, and examined in a RCA EMU-3F transmission electron microscope (8).
RESULTS Epithelial Surface. T h e e p i t h e l i a l s u r f a c e lining t h e g a s t r i c l u m e n c o n s i s t e d o f a single l a y e r o f m u c u s - s e c r e t i n g c e l l s b y L M ( F i g u r e 1A). T h e a p i c e s o f n o r m a l s u r f a c e cells a r e s e e n a s s m o o t h c o n v e x i t i e s , g i v i n g a c o b b l e s t o n e a p p e a r a n c e to t h e m u c o s a l s u r f a c e b y S E M ( F i g u r e IB). I n c r e a s e d m a g n i f i c a t i o n r e v e a l e d t h a t s u r f a c e m i c r o v i l l i on Digestive Diseases, Vol. 23, No. 5 (May 1978)
GASTRIC MUCOSAL ULTRASTRUCTURE
Fig 2. Higher magnification of surface epithelial cells showing the microvilli. (x5000)
SEM (Figure 2) correspond in appearance to those examined with the transmission electron microscope (Figure 3). Gastric pits a p p e a r e d as invaginations in the surface. Occasional surface ceils lying in areas between gastric pits were observed which appeared dead or dying. These cells showed variable types of damage, with nuclei undergoing karyolysis or pyknosis, and cytoplasm having altered staining characteristics (Figure 4). Both the apical cell membrane and nuclear membrane often appeared disrupted in these cells, and there was sloughage of the apical mucous granules. These apparently dying cells show a similar morphology to that seen in cells damaged by aspirin in previous studies (8). One thousand consecutive surface epithelial ceils were counted in each of 12 samples with the light microscope to determine the percent of damaged cells in a normal specimen. This percentage ranged from 1.6% to 3.1% (mean 2.4 _+ 0.2 SE).
Overview of General Structure. When the mucosal surface was broken by blunt dissection, a threedimensional view of the gastric glands underlying the surface epithelial layer was obtained (Figure 5). The gastric pits descend from the surface and are continuous with the underlying gastric glands. Gastric Pits. Foveolae, as viewed from the interior of the mucosa with the scanning electron microscope, appear as tubular structures (Figure 6). The gastric pits are continuous with the gastric glands. Gastric Glands. The gastric glands appeared as branching tubules (Figure 7) with many knoblike projections (Figure 8). The projections correspond to parietal cells which are heavily concentrated in this specific region of the gastric glands when viewed by light microscopy; this is well seen in the LM of the duplicate samples shown in Figure 9. Mucous Epithelial Cells. Mucus was often observed in the lumina of the gastric pits and extended
Fig 3. Transmission electron micrograph showing the microvilli. Two tight junctions (arrows) also are shown. (x41,000) Digestive Diseases, Vol. 23, No. 5 (May 1978)
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Fig 4. Light microscopy section of fundic mucosa, Arrow (A) shows a damaged cell with loss of normal nucleus and abnormal lighter-staining cytoplasm. Arrow (B) illustrates another type of cell damage, with pyknotic nucleus and marked cytoplasm condensation. Note cell (C) with loss of apical granules but intact membrane and nucleus. This cell is considered to represent partial physiologic degranulation and was not counted as a damaged cell. (•
over the surface epithelial cells (Figures 10 and 11). By freeze fracturing the surface epithelial layer the internal structure of the mucous cells could be examined by the scanning electron microscope. Irregular granular structures less than 1 /zm in diameter were seen near the apical surface of the cells (Fig-
ures 10, 11, and 12), which corresponded to mucous granules observed with the light microscope. Parietal Cells. Parietal cells viewed by the scanning electron m i c r o s c o p e after f r e e z e fracture showed canaliculi opening into the glandular lumen (Figure 13). The presence of a moderate number of
Fig. 5. Partially dissected specimen. The surface epithelium (E) is seen with the gastric pits (P). Below these are the gastric glands (arrows). (•
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Fig 6. The surface epithelium and gastric pits (P) are observed from underneath with the lamina propria removed. The base of one pit (arrow) shows an opening that represents the area of continuation into the glands which have been dissected away. (x650)
Fig 7. Fundic glands (FG) are seen as tubular structures with multiple branches (arrows.) (x650) Digestive Diseases, VoL 23, No. 5 (May 1978)
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Fig 8. Increased magnification of the external surface of fundic gland near the neck region. The knoblike structures represent parietal cells (P) viewed from the exterior surface. (x 1500)
Fig 9. Light microscopy through the neck region of fundic glands. Parietal cells (arrows) again are seen bulging from the gland into the lamina propria. (x400)
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Fig 10' ~rac[l~red specimen of the surface lining epithelium. A large strand of mucus (M) ]i~8 in the lumen. Secretory mucous granules can be seen in the apices of the suffor ~pithelial cells. (•
Fig 11. Increased magnification of the apical area of surface epithelial cells. The lateral cell membrane (arrows) of one cell is seen. On either side of this cell are cells that are fractured centrally showing the secretory granules. Microvilli can be seen projecting into the lumen which contains the mucus strand. (x7000) Digestive Diseases, Vol. 23, No. 5 (May 1978)
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Fig 12. Fractured apices of two adjacent cells showing secretory granules. (• 12,000)
Fig 13. Fracture through a parietal cell. The canaliculi (C) can be clearly seen opening into the gastric lumen (L). (x8000)
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Fig 14. A fracture through the base of a gastric gland revealing the apices of several chief cells. There are numerous microvilli projecting into the lumen (L). Zymogen granules also are shown (arrows). (x5000)
microvilli corresponds with the moderate amount of secretion stimulated by the saline meal (13, 14). The mean pH of the saline solution instilled at the beginning of our studies was 5.5. This dropped to 3.6 at the end of 30 rain, consistent with a small increase in acid secretion. Chief Cells. Scanning electron microscopy of fractured chief cells located at the base of the gastric glands d e m o n s t r a t e d intracellular granules throughout the cytoplasm (Figure 14) as compared to the apical location of granules in the surface epithelial cells (7) (Figure 15). The arrangement of chief cells in the base of the gland and the location of zymogen granules within these cells is shown on the LM preparation (Figure 15) of a duplicate biopsy through the same area as Figure 14. DISCUSSION The surface of human gastric mucosa has a cobblestone appearance on scanning electron microscopy. Blunt dissection through this surface permits a three-dimensional view of the gastric pits and glands underneath. Gastric pits have a tubular appearance and the underlying glands connected to these pits branch early in their course. Individual parietal cells in the glands appear as knoblike proDigestive Diseases, Vol. 23, No. 5 (May 1978)
jections when viewed from the external surface. This appearance of parietal cells is similar to that seen in animal studies and correlated well with the position of the parietal cells as observed with the light microscope and transmission electron microscope (9). Scanning electron microscopy followed by blunt dissection and freeze fracture provides a detailed three-dimensional view of the gastric epithelial surface, pits, and glands. Our results definitely show that the gastric glands of man are of a very complex nature, exhibiting numerous branching tubules, in comparison to rodents such as the golden hamster, in which case they are simple tubular structures (9). This technique could be of further value in the examination of abnormal gastric mucosa in disease states such as pernicious anemia, chronic gastritis, and rheumatoid arthritis, which have marked alterations of gastric mucosal morphology and parietal cell mass. Light microscopic examination of the surface epithelium reveals the presence of dying cells in the normal mucosa. The number of abnormal cells is estimated to be between 2% and 3%. Because the majority of the abnormal cells lie in the areas between gastric pits and constitute a predictable population, they are thought to occur normally. Since
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ET AL
Fig 15. Light microscopy through the base of gastric glands. The chief cells (arrows) can be seen surrounding the gland lumen. (x400)
the granules are lost and the cell membrane and nuclear membrane are often disrupted, they are considered to represent abnormal cells rather than cells at different physiological states. They presumably represent surface epithelial cells immediately prior to sloughing. Abnormal cells such as these are also present in normal rat gastric mucosa (15). These observations should be taken into account in studies of the damaging effects of drugs on gastric mucosa (5, 8, 15) so that the percentage of cells damaged can be accurately counted against "normal."
REFERENCES 1. Hingson D J, Ito S: Effect of aspirin and related compounds on the fine structure of mouse gastric mucosa. Gastroenterol0gy 61:156-177, 1971 2. Eastwood GL, Kirchner JP: Changes in the fine structure of mouse gastric epithelium produced by ethanol and urea. Gastroenterology 67:71-84, 1974 3. Hahn K-J, Krischkofski D, Weber E, Morgenstern E: Morphology of gastrointestinal effects of aspirin. Clin Pharmacol Ther 17:330-338, 1975 4. Eastwood GL: Effect of pH on bile salt injury to mouse gastric mucosa. A light and electron microscopic study. Gastroenterology 68:1456-1465, 1975
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5. Ivey KJ, Settree P, Gemmell R: Comparison of acetamirlophen and aspirin on gastric mucosal barrier in man. Correlation with ultrastructural studies. Gastroenterology 68:918, 1975 6. Pfeiffer CJ: Surface topology of the stomach in man and the laboratory ferret. J Ultrastruc Res 33:252-262, 1970 7. Frenning B, OBrink KJ: The effect of acetic and acetylsalicylic acids on the appearance of the gastric mucosal surface epithelium in the scanning electron microscope. Scand J Gastroenterol 6:605-612, 1971 8. Baskin WN, Ivey KJ, Krause WJ, Jeffrey GE, Gemmell RT: Aspirin-induced ultrastructural changes in human gastric mucosa: Correlation with potential difference. Ann Intern Med 85:299-303, 1976 9. Hattori T: On cell proliferation and differentiation of the fundic mucosa of the golden hamster. Fractographic study combined with microscopy and ZH-thymidine autoradiography. Cell Tissue Res 148:213-226, 1974 10. Hattori T, Fugita S: Fractographic study on the growth and multiplication of the gastric gland of the hamster. The gland division cycle. Cell Tissue Res 153:145-149, 1974 11. Miller MM, Revel J-P: Scanning electron triicroscopy of epithelia prepared by blunt dissection. Anat Rec 183!339-358, 1975 12. Boyde A: A method for the preparation of cell surfaces hidden within bulk tissue for examination in the SEM. Scanning Electron Microsc IITRI/1975:296-304, 1975 13. Ito S, Schofield, GC: Studies on the depletion and aceumulaDigestive Diseases, Vol. 23, No. 5 (May 1978)
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tion of microvilli and changes in the tubulovesicular compartment of mouse parietal ceils in relation to gastric acid secretion. J Cell Biol 63:364-382, 1974 14, Frexinos J, Carballido M, Louis A, Rlbet A: Effect of pentagastrin stimulation on human parietal cells. An electron
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microscopic study with quantitative evaluation of cytoplasmic structures. Am J Dig Dis 165:1065-1074, 1971 15. Harding RK, Morris GP: Normal and stress-induced modes of gastric surface epithelial cell (SEC) loss. Gastroenterology 70:970, 1976
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Biopsies from the fundic mucosa of healthy volunteers were examined by scanning electron microscopy following blunt dissection and freeze fracture. The mucosal smface exhibited a cobblestone appearance. With increased magnification, microvilli could be demonstrated on the luminal surface of individual surface cells. Blunt dissection of the gastric mucosa revealed tubular gastric pits descending from the surface until they opened into branched tubular gastric glands. The gastric glands are irregular in outline due to the knoblike projections of individual parietal cells. The internal structure of the component cells also was examined with the scanning electron microscope following freeze fracture. Mucous granules were observed in the apical portion of surface epithelial cells. Lumina of parietal cell canaliculi were found to be continuous with the lumen of the gastric glands. Pepsinogen granules could be seen throughout the cytoplasm of chief cells. The blunt dissection-freeze fracture technique utilizing the scanning electron microscope allows, for the first time, a three-dimensional view of human gastric mucosa, including the gastric pits and glands as well as some of the internal architecture of component cells.
C h a n g e s in g a s t r i c m u c o s a l u l t r a s t r u c t u r e h a v e been increasingly used to study pathological mucosa and the harmful effects of drugs on normal mucosa (1-8). Light microscopy (LM) and transmission electron m i c r o s c o p y (TEM) have been the m a i n s t a y o f s u c h s t u d i e s in b o t h a n i m a l s and man (1-5). More recently, scanning electron mic r o s c o p y (SEM) has added a new dimension to the demonstration of changes in surface epithelium (68). N e w techniques have now been developed to allow the examination of subepithelial structures and
cellular components with the SEM, by fracturing t h e t i s s u e or b y p r e p a r i n g it with b l u n t dissection (9-12). We have used these techniques to examine directly the three-dimensional structure of the human gastric mucosa. Previous structural concepts have been derived from reconstruction of serial sections studied by light and transmission electron microscopy.
From the Departments of Medicine and Anatomy, Hany S. Truman Memorial Veterans Hospital, and University of Missouri, Columbia, Missouri. This work was supported in part by the Medical Research Service of the Veterans Administration and grants from Smith, Kline and French Laboratories, as well as the patient study facilities of the UMMC Clinical Research Center. The technical assistance of Mr. G. E. Jeffrey is gratefully acknowledged. Address for reprint requests: Dr. K. J. Ivey, Division of Gastroenterology, Department of Medicine, University of Missouri Medical Center, Columbia, Missouri 65201.
Healthy male volunteers, aged 20-24 years, were studied after obtaining informed consent and institutional approval from the Human Experimentation Committee. After a 12-hr overnight fast, a nasogastric tube was passed and gastric contents aspirated. The stomach was then lavaged with 100 cc of 150 mM NaC1. A Quinton hydraulic multibiopsy tube was passed orally and positioned fluoroscopically in the gastric fundus. Biopsies, measuring 2 mm x 2 mm, were flushed from the stomach using tyrode solution under COz pressure. Duplicate biopsies were
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Digestive Diseases, Vol. 23, No. 5 (May 1978) 0002-9211/78/0500-0449505.00/1 9 1978 Digestive Disease Systems, Inc.
M A C K E R C H E R ET A L
Fig 1. (A) Light microscopy section of fundic mucosa. The apices of the cells are filled with dark staining mucous granules, and have a convex surface corresponding to "cobblestone effect" of SEM in Figure lB. (x500). (B) Scanning electron microscopy of gastric mucosa. Gastric pits (P) are seen and the surface epithelial cells give a cobblestone appearance. (z500).
taken from the same tube location, within seconds of each other (8). Tissues for SEM were fixed for 4 hr at 0~ C in 3.5% gluteraldehyde buffered in 0.1 M phosphate to pH 7.4. After washing in buffer, they were postfixed in 1% osmium tetroxide at 0 ~ C for 2 hr. The tissues were then blunt-dissected using a scalpel blade to cut one edge of the biopsy. The sides of the incision were then grasped with forceps and the biopsy carefully torn apart to demonstrate the gastric pits and gastric glands prior to critical-point drying, in a fashion similar to that of Hattori (9, 10) and Miller and Revel (11). The tissues were then dehydrated in alcohol and transferred to amyl acetate prior to critical point drying in liquid CO2. Additional tissues after similar fixation procedures were transferred through graded concentrations of ethanol to 100% ethanol, then dropped in a small container filled with liquid nitrogen to fractl~re the specimen for observation of intracellular structures (12). The fractured specimens were critical-point dried by the same method as the blunt-dissected specimens. The dried tissue was then placed on spinner stubs and coated with gold palladium alloy to a depth of 200 A in a vacuum evaporator and viewed with a Cambridge Stereoscope Mark II electron microscope.
450
Duplicate tissue samples were initially fixed for light microscopy and transmission electron microscopy, as were the specimens for scanning electron microscopy. After fixing or initial preparation, tissues were washed twice in 0.1 M phosphate buffer and processed for embedding in Epon 812. Thick sections (1-3/zm) of this material cut for light microscopy were stained in a 1 : 1 solution of 1% toluidine blue and 1% sodium borate. The sections were cut along the greatest length of the specimen. Ultrathin sections of the Epon embedded material cut for elect r o n m i c r o s c o p y w e r e m o u n t e d on u n c o a t e d g r i d s , stained with uranyl acetate and lead citrate, and examined in a RCA EMU-3F transmission electron microscope (8).
RESULTS Epithelial Surface. T h e e p i t h e l i a l s u r f a c e lining t h e g a s t r i c l u m e n c o n s i s t e d o f a single l a y e r o f m u c u s - s e c r e t i n g c e l l s b y L M ( F i g u r e 1A). T h e a p i c e s o f n o r m a l s u r f a c e cells a r e s e e n a s s m o o t h c o n v e x i t i e s , g i v i n g a c o b b l e s t o n e a p p e a r a n c e to t h e m u c o s a l s u r f a c e b y S E M ( F i g u r e IB). I n c r e a s e d m a g n i f i c a t i o n r e v e a l e d t h a t s u r f a c e m i c r o v i l l i on Digestive Diseases, Vol. 23, No. 5 (May 1978)
GASTRIC MUCOSAL ULTRASTRUCTURE
Fig 2. Higher magnification of surface epithelial cells showing the microvilli. (x5000)
SEM (Figure 2) correspond in appearance to those examined with the transmission electron microscope (Figure 3). Gastric pits a p p e a r e d as invaginations in the surface. Occasional surface ceils lying in areas between gastric pits were observed which appeared dead or dying. These cells showed variable types of damage, with nuclei undergoing karyolysis or pyknosis, and cytoplasm having altered staining characteristics (Figure 4). Both the apical cell membrane and nuclear membrane often appeared disrupted in these cells, and there was sloughage of the apical mucous granules. These apparently dying cells show a similar morphology to that seen in cells damaged by aspirin in previous studies (8). One thousand consecutive surface epithelial ceils were counted in each of 12 samples with the light microscope to determine the percent of damaged cells in a normal specimen. This percentage ranged from 1.6% to 3.1% (mean 2.4 _+ 0.2 SE).
Overview of General Structure. When the mucosal surface was broken by blunt dissection, a threedimensional view of the gastric glands underlying the surface epithelial layer was obtained (Figure 5). The gastric pits descend from the surface and are continuous with the underlying gastric glands. Gastric Pits. Foveolae, as viewed from the interior of the mucosa with the scanning electron microscope, appear as tubular structures (Figure 6). The gastric pits are continuous with the gastric glands. Gastric Glands. The gastric glands appeared as branching tubules (Figure 7) with many knoblike projections (Figure 8). The projections correspond to parietal cells which are heavily concentrated in this specific region of the gastric glands when viewed by light microscopy; this is well seen in the LM of the duplicate samples shown in Figure 9. Mucous Epithelial Cells. Mucus was often observed in the lumina of the gastric pits and extended
Fig 3. Transmission electron micrograph showing the microvilli. Two tight junctions (arrows) also are shown. (x41,000) Digestive Diseases, Vol. 23, No. 5 (May 1978)
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Fig 4. Light microscopy section of fundic mucosa, Arrow (A) shows a damaged cell with loss of normal nucleus and abnormal lighter-staining cytoplasm. Arrow (B) illustrates another type of cell damage, with pyknotic nucleus and marked cytoplasm condensation. Note cell (C) with loss of apical granules but intact membrane and nucleus. This cell is considered to represent partial physiologic degranulation and was not counted as a damaged cell. (•
over the surface epithelial cells (Figures 10 and 11). By freeze fracturing the surface epithelial layer the internal structure of the mucous cells could be examined by the scanning electron microscope. Irregular granular structures less than 1 /zm in diameter were seen near the apical surface of the cells (Fig-
ures 10, 11, and 12), which corresponded to mucous granules observed with the light microscope. Parietal Cells. Parietal cells viewed by the scanning electron m i c r o s c o p e after f r e e z e fracture showed canaliculi opening into the glandular lumen (Figure 13). The presence of a moderate number of
Fig. 5. Partially dissected specimen. The surface epithelium (E) is seen with the gastric pits (P). Below these are the gastric glands (arrows). (•
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Fig 6. The surface epithelium and gastric pits (P) are observed from underneath with the lamina propria removed. The base of one pit (arrow) shows an opening that represents the area of continuation into the glands which have been dissected away. (x650)
Fig 7. Fundic glands (FG) are seen as tubular structures with multiple branches (arrows.) (x650) Digestive Diseases, VoL 23, No. 5 (May 1978)
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Fig 8. Increased magnification of the external surface of fundic gland near the neck region. The knoblike structures represent parietal cells (P) viewed from the exterior surface. (x 1500)
Fig 9. Light microscopy through the neck region of fundic glands. Parietal cells (arrows) again are seen bulging from the gland into the lamina propria. (x400)
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Fig 10' ~rac[l~red specimen of the surface lining epithelium. A large strand of mucus (M) ]i~8 in the lumen. Secretory mucous granules can be seen in the apices of the suffor ~pithelial cells. (•
Fig 11. Increased magnification of the apical area of surface epithelial cells. The lateral cell membrane (arrows) of one cell is seen. On either side of this cell are cells that are fractured centrally showing the secretory granules. Microvilli can be seen projecting into the lumen which contains the mucus strand. (x7000) Digestive Diseases, Vol. 23, No. 5 (May 1978)
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Fig 12. Fractured apices of two adjacent cells showing secretory granules. (• 12,000)
Fig 13. Fracture through a parietal cell. The canaliculi (C) can be clearly seen opening into the gastric lumen (L). (x8000)
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Fig 14. A fracture through the base of a gastric gland revealing the apices of several chief cells. There are numerous microvilli projecting into the lumen (L). Zymogen granules also are shown (arrows). (x5000)
microvilli corresponds with the moderate amount of secretion stimulated by the saline meal (13, 14). The mean pH of the saline solution instilled at the beginning of our studies was 5.5. This dropped to 3.6 at the end of 30 rain, consistent with a small increase in acid secretion. Chief Cells. Scanning electron microscopy of fractured chief cells located at the base of the gastric glands d e m o n s t r a t e d intracellular granules throughout the cytoplasm (Figure 14) as compared to the apical location of granules in the surface epithelial cells (7) (Figure 15). The arrangement of chief cells in the base of the gland and the location of zymogen granules within these cells is shown on the LM preparation (Figure 15) of a duplicate biopsy through the same area as Figure 14. DISCUSSION The surface of human gastric mucosa has a cobblestone appearance on scanning electron microscopy. Blunt dissection through this surface permits a three-dimensional view of the gastric pits and glands underneath. Gastric pits have a tubular appearance and the underlying glands connected to these pits branch early in their course. Individual parietal cells in the glands appear as knoblike proDigestive Diseases, Vol. 23, No. 5 (May 1978)
jections when viewed from the external surface. This appearance of parietal cells is similar to that seen in animal studies and correlated well with the position of the parietal cells as observed with the light microscope and transmission electron microscope (9). Scanning electron microscopy followed by blunt dissection and freeze fracture provides a detailed three-dimensional view of the gastric epithelial surface, pits, and glands. Our results definitely show that the gastric glands of man are of a very complex nature, exhibiting numerous branching tubules, in comparison to rodents such as the golden hamster, in which case they are simple tubular structures (9). This technique could be of further value in the examination of abnormal gastric mucosa in disease states such as pernicious anemia, chronic gastritis, and rheumatoid arthritis, which have marked alterations of gastric mucosal morphology and parietal cell mass. Light microscopic examination of the surface epithelium reveals the presence of dying cells in the normal mucosa. The number of abnormal cells is estimated to be between 2% and 3%. Because the majority of the abnormal cells lie in the areas between gastric pits and constitute a predictable population, they are thought to occur normally. Since
457
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ET AL
Fig 15. Light microscopy through the base of gastric glands. The chief cells (arrows) can be seen surrounding the gland lumen. (x400)
the granules are lost and the cell membrane and nuclear membrane are often disrupted, they are considered to represent abnormal cells rather than cells at different physiological states. They presumably represent surface epithelial cells immediately prior to sloughing. Abnormal cells such as these are also present in normal rat gastric mucosa (15). These observations should be taken into account in studies of the damaging effects of drugs on gastric mucosa (5, 8, 15) so that the percentage of cells damaged can be accurately counted against "normal."
REFERENCES 1. Hingson D J, Ito S: Effect of aspirin and related compounds on the fine structure of mouse gastric mucosa. Gastroenterol0gy 61:156-177, 1971 2. Eastwood GL, Kirchner JP: Changes in the fine structure of mouse gastric epithelium produced by ethanol and urea. Gastroenterology 67:71-84, 1974 3. Hahn K-J, Krischkofski D, Weber E, Morgenstern E: Morphology of gastrointestinal effects of aspirin. Clin Pharmacol Ther 17:330-338, 1975 4. Eastwood GL: Effect of pH on bile salt injury to mouse gastric mucosa. A light and electron microscopic study. Gastroenterology 68:1456-1465, 1975
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