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Scanning electron microscopy of gastroscopic biopsies
Scanning electron microscopy of gastroscopic biopsies Ebrahim Fallah, MD Bernard M. Schuman, MD John H. L. Watson, PhD Jessica Goodwin, BS Detroit, Michigan
Endoscopic biopsy specimens of normal human gastric mucosa and mucosa in erosive and chronic gastritis were studied by scanning electron microscopy. The orifices of the gastric pits and the cobblestone surface of epithelial cells covered with villus-like and bulbous projections were observed. A possible mechanism for mucus secretion from these cells is suggested. The mucosa showed striking morphologic surface differences from the normal in cases of both erosive and chronic gastritis.
T
he gastric mucosa is unique among living surfaces for it must cope with and maintain its integrity under a wide variety of environmental conditions. It is affected by temperature, osmolality, mechanical trauma and differing ingested substances, including both food and drugs. Its inherent ability to withstand the action of hydrochloric acid and proteolytic enzymes still remains a mystery. In addition, the exact mechanisms responsible for gastric mucosal injury in various pathologic states are not yet fully understood 102 , although it is known that the gastric epithelium plays a major role in the formation of the so-called mucosal barrier. 3 We have employed the scanning electron microscope in studies of gastroscopic biopsies as an aid toward the elucidation of the nature and structure of this unique mucosal surface and in an attempt to understand more fully its alterations to injury.
MATERIALS AND METHODS Forceps biopsies of gastric mucosa were obtained from 4 normal human stomachs, from 3 patients with erosive gastritis, and from 3 with chronic non-specific gastritis. The cases were selected from among patients referred to the gastrointestinal endoscopy unit for evaluation of a wide variety of conditions. Endoscopic diagnosis was used for the initial classification which was later confirmed by light microscopy'. Three biopsies with an average diameter of 1 mm were taken in each case: 2 from the body and 1 from the antrum of each stomach. One body biopsy was paraffin-sectioned and H&E stained for conventional light microscopy. The other and the antrum biopsy were put immediately into cacodylate-buffered, 2%
glutaraldehyde solution for fixation. One half of each of these was critical-point dried in C02 and subsequently vacuumcoated with gold-palladium for scanning electron microscopy. The other half of each was processed for transmission electron microscopy by conventional methods and without critical point drying, using Araldite for embedment. In addition, several of the critical-point dried specimens were reprocessed for transmission after examination by scanning electron microscopy. It has long been known that shortening the exposure time to outside environment by immediate fixation of the tissue as it leaves the donor reduces autolytic processes to a minimum, and preserves the surface as closely as possible in its in vivo condition. Concurrent experiments to determine the effects of washing and other preparative techniques upon canine stomach mucosaS (e.g., rinsing in normal saline, ice water, or 1% HCI), showed that any treatment or manipulation before fixation is capable of introducing surface alterations. Consequently, every sample examined in this study was immediately immersed in the fixative without pretreatment of any kind. It was found, when the specimen surface was covered by an original thick layer of mucus after fixation, the mucus could be carefully teased from the surface mechanically, leaving the cell surface undamaged. The antrum biopsies were examined for each case by scanning and for most by transmission electron microscopy without noting any surface differences relative to the specimens taken from the body of the same stomach. Consequently all of the following report concerns the body of the stomach.
From the Division of Gastroenterology, Henry Ford Hospital, and the Department of Physics and Biophysics, Edsel B. Ford Institute for Medical Research, Detroit, Michigan. Reprint requests: John H. L. Watson, PhD, Department of Physics and Biophysics, Edsel B. Ford Institute for Medical Research, 2799 West Grand Blvd., Detroit, Michigan 48202. VOLUME 22, NO.3, 1976
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GASTROINTESTINAL ENDOSCOPY
Although it is always difficult and usually speculative to draw dynamic conclusions from the 2-dimensional images of static electron micrographs, what could be all stages in the formation of these projections by an extrusion process from the cell cytoplasm can be visualized in Figures 3 and 4, from a first nippl ing of the epithelial surface, through an intermediate stage of larger maturing "particles", to the final end-product of spherical "particles" raised well above the surface. While some of the properties of these bulbous structures are similar to those of microvilli (they are of similar diameter and by transmission electron microscopy of thin sections sometimes have a root-like structure) (as in Figure 14A), neither their peculiar shape, short length, nor the apparent process of their formation are usual characteristics of microvilli. Many of the projections are shaped like thumbs (clear arrow, Figure 4,) abou~ 1,5% wider at their rounded extremity than at their base but without a narrow supporting stalk. As such they resemble true microvilli more closely, but are still connected laterally in <:l nonmicrovillus fashion by short, thick necks. There is also evidence that as the rounded "particles" are extruded they draw the lateral necks and stalks with them through the surface (Figures 3,4). This strange phenomenon is also observed by transmission (Figure 4E). In Figure 5, at the arrowhead, 5 stalks are emerging from the epithelium in a single area. In the normal human stomach (Figure I), the openings to gastric pits (diameters 3 to 15 microns), are round or oval and located relatively uniformly over the surface (one pit per 3,500 square microns). At low magnification the surface is relatively smooth, with a mottled appearance, and debris is observed scattered over the surface. At higher power (Figure 2) the cells are seen to be slightly raised, giving a cobblestone appearance to the surface, and over the cells there is a dispersion of tiny depressions or "micropits" (mP) of unknown nature and function. At the increased magnification the appearance of mottling is found to be a result of a surface population of unique "bulb-like structures" which tend to concentrate in normal stomachs toward the margins of the cells. The spherical ends of these projections, which at this magnification and angle appear as round "particles" of diameter about 250 nm, *, outlining cell margins, are seen at even higher power in Figure 3. The "particles" are observed to be connected laterally in a "tinker toy" fashion by short, stubby necks approximately 100 nm thick, running parallel to the cell surface in a variety of directions. Occasionally it is also possible to observe that some of the "particles" are connected to the cell surface itself by short stalks of the same diameter. The viewing angle is not often optimum for showing this, but examples are marked at the arrowheads in Figure 3. Figure 3A is an enlarged view of part of Figure 3, to show the stalks more clearly. At the clear arrow in Figure 3, a hole in the surface of the cell is marked where such a stalk has probably been torn away. *1 nm = 1 nanometer = 10 Angstrom units = 10-' em
Scanning electron microscopy of the surface of normal stomach has led to a further observation which may explain the mechanism by which mucus granules within the cytoplasm are secreted into the lumen. In Figure 5, at (G) there is a wide opening in the mucosa through which granules are emerging. Their sizes are consistent with those of the mucus granules which are observed to exist just under the epithelial membrane by transmission electron microscopy of gastric mucosa. Mucus granules penetrating the surface are also demonstrable by transmission. The frequent lumpiness of the surface is thought to be partly a function of pressure exerted by these granules as they reach a level just under the cytoplasmic membrane, as in the region (X) in Figure 2. The pressure of the granules against the cytoplasmic membrane increases until at some point the membrane breaks to release the granules into the lumen. The resulting break in the surface then reseals itself. In erosive gastritis (Figure 6) the mucosa was covered in most areas with a large amount of debris and exudate, with some red blood cells from the bleeding. The ostia to the gastric pits were increased in number (1 pit per 2300 square microns), and many were at least partly occluded with the exudate. The cells immediately surrounding the ostia seemed to be swollen and elongated. Cobblestoning appeared to be more prominent in erosive gastritis than in normal mucosa, adding to the impression of lumpiness of the surface. Erosion of the surface was difficult to recognize at low magnification because of the debris covering it but could be recognized focally at higher power (Figure 7). The individual cells are seen to be much smoother than in the normal and to contain very few if any micropits. There was a significant number of large surface blebs, some of which are marked at the arrows in Figure 7. The bulbous projections of the mucosal surface (Figure 8) were again present, as they were in every specimen we have examined, but they were somewhat different than they were in the normal, being smaller in diameter (160 to 240 nm) and somewhat sparser. They tended to be distributed evenly over the cell surfaces rather than concentrated toward the cell margins. The stalks and connecting necks were also of smaller caliber (less than 80 nm). In addition, in gastritic mucosa there was a higher percentage of the stubby thumb-like projections vis-a-vis the bulbous projections. In chronic non-specific gastritis (Figure 9) the surface of the gastric mucosa was extremely coarse and cobblestoned and was completely different from that of either normal mucosa or erosive gastritis. The individual cells varied widely in size and shape and were noticeably raised to present a striking cobblestone appearance. The ostia of the gastric pits were huge and varied widely in both size and shape. They were dilated, often elongated and much reduced in number (1 pit per 23,000 square microns). Particularly near the entrances to the ostia of the pits, there were multiple areas of focal cell degeneration. The epithelial cells are raised so that their margins (Figure 10)
Figures: (1) The surface of normal human stomach mucosa, showing the openings to the gastric pits, a mottled surface and some debris. x390. (In all figures the length of the line marker represents one micron, unless marked otherwise). (2) Part of (1) showing bulbous projections of the mucosa concentrated at cell margins, cobblestone structure of the surface and micropits (mP). All observations have been aided by detailed stereoscopic viewing of the micrographs. At X is a thin region where mucus granules under the surface give it a lumpy appearance. x5,200. (3) Part of (2) at higher magnification to show lateral, connecting necks between bulbous projections of the mucosa. The various stages of the process by which the "particles" could form and extrude from the mucosal surface are also demonstrated. At the arrows is evidence for supporting stalks or connecting necks being drawn up parallel to and through the surface. At the arrowhead is an example of a stalk from the surface supporting a bulbous projection, and at the clear arrow is a hole presumably left when such a stalk was torn away. x24,000. (3A) Detail from (3); atthe arrows, stalks from the surface support bulbous projections of the mucosa. x51,600. VOLUME 22, NO.3, 1976
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were outlined sharply in cobblestone relief. Unlike the normal, the numerous mucosal projections were evenly distributed over the cell surfaces, there were few if any lateral necks between them (Figure 11), and many of the projections rose with uniform thickness to considerable distances above the cell urface, resembling true microvilli, rather than bulbous projections. The tips of the projections were rounded as they were also in the normal and erosive gastritic mucosa.
DISCUSSION The epithelium of normal stomach consists of a single layer of tall, columnar, mucus-secreting cells, "the mucus surface cells". Both their ultrastructure and kinetics have been extensively studied and reported '.7 In an effort to facilitate the diagnosis of gastritis gastric biopsies have been surfaceexamined with the dissecting light microscope 8 - lo . The relatively low resolution of the light microscope has hindered progress in this effort, but interest in surface morphology of gastric mucosa in disease has revived 11-13 since the development of commercially available scanning electron microscopesl 4 with their ability to study biological surfaces directly at improved resolution and depth of field. 14 The present study has demonstrated that the tech niques of specimen preparation for scanning electron microscopy are appl icable to human stomach mucosa derived from endoscopic biopsies and that excellent preservation of ultrastruc-
tures can be achieved. The immediate fixation of the biopsy specimen without pretreatment plus the critical-point drying, as well as the higher resolution of the scanning microscope may explain why the observation of bulb-like projections of the epithelial surfaces have not been reported to our knowledge by other investigators. It is assumed that these projections are not artifactual since they are seen in some form in both normal and pathological specimens, and their shape, distribution, and frequency seem to change from one disease entity to another. Indeed, since critical-point drying is known to preserve the biological structures better than other methods of drying, artifacts could be expected to be minimal in these samples 1S . In addition, since some true microvilli are seen in all cases (the percentage being higher in gastritis) and since both bulbous projections and microvilli are seen in a single field, it is reasonable to conclude that neither structure is an artifact. It has been suggested that the lateral necks could be merely a manifestation of the metal-coating on the fibers of the dried furry coat (Figure 12). Size considerations would favor this explanation as wou Id arguments based on the geometry of the microvilli (Mv) and the furry coat in thin section. On the other hand, other observations would argue against such an explanation, particularly the great length of many of the necks and thei r observed tendency to re-enter and to re-emerge linearly from the cytoplasm (Figure 13). What seems to be a continu-
Figures: (4) Another part of (2) showing the same features as (3) and examples of thumb-like projections in the region of the clear arrows. At the solid arrows are further examples of necks apparently being pulled up parallel to and through the mucosal surface by the extruding "particles". x24,000. (5) An area of normal mucosa, showing mucus granules (e) penetrating into the lumen through an open area of the mucosa. Further examples of micropits (mP) and of stalks emerging from the surface are marked in the region of the arrowhead. xl 0,000. 140
GASTROINTESTINAL ENDOSCOPY
Figures (6) The surface of the mucosa in erosive gastritis, showing gastric pits; there is much debris with some red blood cells. x61 O. (7) Part of (6) with blebs marked at the arrows, and an area of focal erosion at the clear arrows. x4, 800. (8) Part of (7) where the bulbous projections are smaller than in the normal and are joined by necks of decreased diameter. What appears to be a long, continuous linearity joining "particle" to "particle" and emerging from and reentering the mucosal surface is marked by the arrows. x 12,000. VOLUME 22, NO.3, 1976
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Figures (9) The surface of the mucosa in chronic non-specific gastritis. Regions of focal cell degeneration are marked at the arrows. x31O. (10) Part of (9) where the cobblestone relief is very prominent. The mucosal projections are evenly distributed over the surface of the cells, and many of them are much longer and more microvillus-like than the bulbous projections described for the normal cells. x3,200. (11) Part of (1 0) where almost all of the mucosal projections appear to be thumb-like projections or true microvilli. They are distributed fairly evenly over the individual cel/surfaces, and are not joined together by necks. x 77,500. 142
GASTROINTESTINAL ENDOSCOPY
Figures: (12) A transmission electron micrograph of an isolated, heavily microvilliated area of normal human stomach mucosa, showing the microvilli in approximate cross section with the fibers of the furry coat between them. Since true microvilli as well as bulbous projections can be found in every specimen, we conclude that some components of the surface structure as seen by scanning are usually microvilli. x 36,000 (13) Normal human stomach mucosa showing micropits (mP) and, at the arrows, what appears to be a continuous rod-like neck, which connects many "particles" together, and emerges from and reenters the mucosal surface many times in the process. xIB,BOO.
ous single neck (marked at the arrows in Figure 13) enters, emerges from, and re-enters the cytoplasm, connecting a number of "particles" together as it does so. The same phenomenon is observable in mucosa in erosive gastritis (Figure 8) . Certainly, the complete nature, composition, and function of these very puzzling ultrastructures remains unexplained. In order to study the bulbous projections in cross section and at higher resolution, 2 of the specimens which had been critical-point dried, coated with gold-palladium, and examined by scanning, were re-embedded in Araldite and thin sectioned for transmission electron microscopy. It has not been possible to find fields by transmission which demonstrate the stalks and necks clearly or explain their presence, but evidence has been found in the micrographs for all of the observations reported from scanning the same sample (Figure 14). The bulbous endings were found to be membrane-bound and connected to the cell membrane by stalks and to each other by metal-coated necks. Frequently, it appeared as if a round granule (diameter 150 to 200 nm) was contained in the bulbous end. Pffeifer"'12 and others have reported the presence of microvilli in human gastric mucosa. The bulbous projections observed in addition to microvilli in the present study have the same diameter as microvilli but are somewhat shorter in length and have a different shape. The bulbous ends, supporting stalks and lateral necks of uniform caliber but VOLUME 22, NO.3, 1976
varied lengths, are not characteristics of microvilli. Nor do the lateral necks seem to be liquid or to form in any way at the expense of the bulbous bodies which they join together. Finally, the projections themselves seem to form by extrusion from the cell surface in a manner which is unlike that expected for microvilli.
Figure 14. Transmission electron micrographs of critical-point dried, gold-palladium coated, normal mucosa reembedded in Araldite after scanning, and thin sectioned. Observations of supporting stalks, joining necks, extrusion from the surface, round granules within the extremities of the bulbous projections (clear arrows), roots on some of the projections (arrow), and necks or stalks (arrowhead) being drawn up through the surface are demonstrated. x39,000. 143
Figure 15. Normal human stomach mucosa, critical-point dried and coated with goldpalladium for scanning microscopy. A region of mucosal projections and joining necks is seen to form a web over the surface. x20,OOO. Speculation about the possible function of these projections is even more hazardous than hypothesis concerning their structure. Andrew and Porter 16 have reported microvilli on the surface of mesothelial cells, proposing that they might function as a holding grid to entrap mucus and reduce friction against serosal surfaces. If the same entrapment mechanism could operate in the stomach, albeit for a different purpose, one can speculate that these projections with their interconnecting necks could eventually form a web of knotted strands to create a protective layer over the mucosal surface 17 • This could entrap secreted mucus which, after undergoing rheological changes in the new environment, could form a sort of protective membrane that contributes to mucosal resistance. That such webs do exist on the surface of normal mucosa is illustrated in Figure 15. However, complete substantiation of this would require a better understanding of both the composition and function of gastric mucus's, as well as of its interaction with cell membranes and with these projections. It is not yet possible to conclude that these projections, Iike microvilli
in the small bowel, play any part in selective absorption from the stomach. The vast increase in the numbers of true microvilli in gastritis, especially chronic, seen by both transmission and scanning electron microscopy, tends to indicate that the cells are reacting to a stimulus. It also indicates, along with their similarity in appearance, that the bulbous projections are a special form of microvilli and consequently that the microvilli and the bulbous projections form by the same extrusion process. Striking differences in surface morphology have been observed in the present study between the normal and gastritic mucosa. Because of the limited number of cases, the significance of these variations requires further investigation. Biological staining and biochemical analysis of surface structures combined with scanning and transmission electron microscopy of gastroscopic biopsies might further clarify the bioengineering design of gastric mucosa.
REFERENCES
evidence for in situ surface cell degeneration. Exp Mol Patho/13:19, 1970 12. PFEIFFER CI. GEOFFREY R, JOSEF W: Gastrointestinal ultrastructure, in, An Atlas of Scanning and Transmission Electron Micrographs, Academic Press, New York and London, 1974 13. KATSU K: Scanni ng electron microscopic study of the gastroi ntesti naltract; 3-dimensional observation of the mucosal surface. lap I Gastroengerology (in japanese) 70:812, 1973 14. WATSON JHL: The scanning electron microscope. Henry Ford HospMedl 21:75,1973 15. POLLIACK A, LAMPEN N, deHARVEN E: Comparison of air drying and critical point drying procedures for the study of human blood cells by scanning electron microscopy. In: IITRIISEM.Ed. johari and Cowin, Chicago, 1973 16. ANDREWS PM, PORTER KR: The ultrastructural morphology and possible functional significance of mesothelial microvilli. Anal Rec 177:409, 1973 17. WATSON JHL, GOODWIN, J, FALLAH E: Some observations on the ultrastructure of the surface of normal human stomach mucosa. In 33rd Ann Proc Electron Microscopy Soc Amer. Ed. GW Bailey. Claitor's Publishing Division, Baton Rouge, 1975 18. SCHRAGER J: The chemical composition and function of gastrointestinal mucus. Gut 11 :450, 1970
1. PALMER ED: Gastritis: a revaluation. Medicine 33:199, 1954 2. WOOD II. TAFT 1I: Diffuse Lesions of the Stomach. Edward Arnold, london, 1958 3. DAVENPORT HW: Back diffusion of acid through the gastric mucosa and its physiologic consequences. In, Progress in Gastroenterology, Vol. 2, GBj Glass, Ed. London, Grune and Stratton, Inc., P. 42, 1970 4. ANDERSON TF : Techniques for the preservation of 3-dimensional structure in preparing specimens for the electron microscope. Tr N Y Acad Sci 13:130, 1951 5. WATSON JHL: Unpublished research, 1974 6. LILLIBRIDGE CB: The fine structure of normal human gastric mucosa. Gastroenterology 47:269, 1964 7. RUBIN W, Ross LL, SLEISENGER MH, JEFFRIES GH: The normal human gastric epithelia. Lab Inv 19:98, 1968 8. BURHOL PG: Surface microscopic and histologic appearances of the gastric mucosa. Scand I Gaslroenterol 3: 17, 1968 9. SALEM SN, TRUELOVE SC: Dissecting microscope appearances of the gastric mucosa. Br Med I 2: 1S03, 1964 10. SALEM SN: Gastric mucosa under the dissecting microscope. Am I Dig Dis 10:705, 1965 11. PFEIFFER CJ: Gastric surface morphology in man, monkey and ferret;
144
GASTROINTESTINAL ENDOSCOPY
Endoscopic biopsy specimens of normal human gastric mucosa and mucosa in erosive and chronic gastritis were studied by scanning electron microscopy. The orifices of the gastric pits and the cobblestone surface of epithelial cells covered with villus-like and bulbous projections were observed. A possible mechanism for mucus secretion from these cells is suggested. The mucosa showed striking morphologic surface differences from the normal in cases of both erosive and chronic gastritis.
T
he gastric mucosa is unique among living surfaces for it must cope with and maintain its integrity under a wide variety of environmental conditions. It is affected by temperature, osmolality, mechanical trauma and differing ingested substances, including both food and drugs. Its inherent ability to withstand the action of hydrochloric acid and proteolytic enzymes still remains a mystery. In addition, the exact mechanisms responsible for gastric mucosal injury in various pathologic states are not yet fully understood 102 , although it is known that the gastric epithelium plays a major role in the formation of the so-called mucosal barrier. 3 We have employed the scanning electron microscope in studies of gastroscopic biopsies as an aid toward the elucidation of the nature and structure of this unique mucosal surface and in an attempt to understand more fully its alterations to injury.
MATERIALS AND METHODS Forceps biopsies of gastric mucosa were obtained from 4 normal human stomachs, from 3 patients with erosive gastritis, and from 3 with chronic non-specific gastritis. The cases were selected from among patients referred to the gastrointestinal endoscopy unit for evaluation of a wide variety of conditions. Endoscopic diagnosis was used for the initial classification which was later confirmed by light microscopy'. Three biopsies with an average diameter of 1 mm were taken in each case: 2 from the body and 1 from the antrum of each stomach. One body biopsy was paraffin-sectioned and H&E stained for conventional light microscopy. The other and the antrum biopsy were put immediately into cacodylate-buffered, 2%
glutaraldehyde solution for fixation. One half of each of these was critical-point dried in C02 and subsequently vacuumcoated with gold-palladium for scanning electron microscopy. The other half of each was processed for transmission electron microscopy by conventional methods and without critical point drying, using Araldite for embedment. In addition, several of the critical-point dried specimens were reprocessed for transmission after examination by scanning electron microscopy. It has long been known that shortening the exposure time to outside environment by immediate fixation of the tissue as it leaves the donor reduces autolytic processes to a minimum, and preserves the surface as closely as possible in its in vivo condition. Concurrent experiments to determine the effects of washing and other preparative techniques upon canine stomach mucosaS (e.g., rinsing in normal saline, ice water, or 1% HCI), showed that any treatment or manipulation before fixation is capable of introducing surface alterations. Consequently, every sample examined in this study was immediately immersed in the fixative without pretreatment of any kind. It was found, when the specimen surface was covered by an original thick layer of mucus after fixation, the mucus could be carefully teased from the surface mechanically, leaving the cell surface undamaged. The antrum biopsies were examined for each case by scanning and for most by transmission electron microscopy without noting any surface differences relative to the specimens taken from the body of the same stomach. Consequently all of the following report concerns the body of the stomach.
From the Division of Gastroenterology, Henry Ford Hospital, and the Department of Physics and Biophysics, Edsel B. Ford Institute for Medical Research, Detroit, Michigan. Reprint requests: John H. L. Watson, PhD, Department of Physics and Biophysics, Edsel B. Ford Institute for Medical Research, 2799 West Grand Blvd., Detroit, Michigan 48202. VOLUME 22, NO.3, 1976
137
138
GASTROINTESTINAL ENDOSCOPY
Although it is always difficult and usually speculative to draw dynamic conclusions from the 2-dimensional images of static electron micrographs, what could be all stages in the formation of these projections by an extrusion process from the cell cytoplasm can be visualized in Figures 3 and 4, from a first nippl ing of the epithelial surface, through an intermediate stage of larger maturing "particles", to the final end-product of spherical "particles" raised well above the surface. While some of the properties of these bulbous structures are similar to those of microvilli (they are of similar diameter and by transmission electron microscopy of thin sections sometimes have a root-like structure) (as in Figure 14A), neither their peculiar shape, short length, nor the apparent process of their formation are usual characteristics of microvilli. Many of the projections are shaped like thumbs (clear arrow, Figure 4,) abou~ 1,5% wider at their rounded extremity than at their base but without a narrow supporting stalk. As such they resemble true microvilli more closely, but are still connected laterally in <:l nonmicrovillus fashion by short, thick necks. There is also evidence that as the rounded "particles" are extruded they draw the lateral necks and stalks with them through the surface (Figures 3,4). This strange phenomenon is also observed by transmission (Figure 4E). In Figure 5, at the arrowhead, 5 stalks are emerging from the epithelium in a single area. In the normal human stomach (Figure I), the openings to gastric pits (diameters 3 to 15 microns), are round or oval and located relatively uniformly over the surface (one pit per 3,500 square microns). At low magnification the surface is relatively smooth, with a mottled appearance, and debris is observed scattered over the surface. At higher power (Figure 2) the cells are seen to be slightly raised, giving a cobblestone appearance to the surface, and over the cells there is a dispersion of tiny depressions or "micropits" (mP) of unknown nature and function. At the increased magnification the appearance of mottling is found to be a result of a surface population of unique "bulb-like structures" which tend to concentrate in normal stomachs toward the margins of the cells. The spherical ends of these projections, which at this magnification and angle appear as round "particles" of diameter about 250 nm, *, outlining cell margins, are seen at even higher power in Figure 3. The "particles" are observed to be connected laterally in a "tinker toy" fashion by short, stubby necks approximately 100 nm thick, running parallel to the cell surface in a variety of directions. Occasionally it is also possible to observe that some of the "particles" are connected to the cell surface itself by short stalks of the same diameter. The viewing angle is not often optimum for showing this, but examples are marked at the arrowheads in Figure 3. Figure 3A is an enlarged view of part of Figure 3, to show the stalks more clearly. At the clear arrow in Figure 3, a hole in the surface of the cell is marked where such a stalk has probably been torn away. *1 nm = 1 nanometer = 10 Angstrom units = 10-' em
Scanning electron microscopy of the surface of normal stomach has led to a further observation which may explain the mechanism by which mucus granules within the cytoplasm are secreted into the lumen. In Figure 5, at (G) there is a wide opening in the mucosa through which granules are emerging. Their sizes are consistent with those of the mucus granules which are observed to exist just under the epithelial membrane by transmission electron microscopy of gastric mucosa. Mucus granules penetrating the surface are also demonstrable by transmission. The frequent lumpiness of the surface is thought to be partly a function of pressure exerted by these granules as they reach a level just under the cytoplasmic membrane, as in the region (X) in Figure 2. The pressure of the granules against the cytoplasmic membrane increases until at some point the membrane breaks to release the granules into the lumen. The resulting break in the surface then reseals itself. In erosive gastritis (Figure 6) the mucosa was covered in most areas with a large amount of debris and exudate, with some red blood cells from the bleeding. The ostia to the gastric pits were increased in number (1 pit per 2300 square microns), and many were at least partly occluded with the exudate. The cells immediately surrounding the ostia seemed to be swollen and elongated. Cobblestoning appeared to be more prominent in erosive gastritis than in normal mucosa, adding to the impression of lumpiness of the surface. Erosion of the surface was difficult to recognize at low magnification because of the debris covering it but could be recognized focally at higher power (Figure 7). The individual cells are seen to be much smoother than in the normal and to contain very few if any micropits. There was a significant number of large surface blebs, some of which are marked at the arrows in Figure 7. The bulbous projections of the mucosal surface (Figure 8) were again present, as they were in every specimen we have examined, but they were somewhat different than they were in the normal, being smaller in diameter (160 to 240 nm) and somewhat sparser. They tended to be distributed evenly over the cell surfaces rather than concentrated toward the cell margins. The stalks and connecting necks were also of smaller caliber (less than 80 nm). In addition, in gastritic mucosa there was a higher percentage of the stubby thumb-like projections vis-a-vis the bulbous projections. In chronic non-specific gastritis (Figure 9) the surface of the gastric mucosa was extremely coarse and cobblestoned and was completely different from that of either normal mucosa or erosive gastritis. The individual cells varied widely in size and shape and were noticeably raised to present a striking cobblestone appearance. The ostia of the gastric pits were huge and varied widely in both size and shape. They were dilated, often elongated and much reduced in number (1 pit per 23,000 square microns). Particularly near the entrances to the ostia of the pits, there were multiple areas of focal cell degeneration. The epithelial cells are raised so that their margins (Figure 10)
Figures: (1) The surface of normal human stomach mucosa, showing the openings to the gastric pits, a mottled surface and some debris. x390. (In all figures the length of the line marker represents one micron, unless marked otherwise). (2) Part of (1) showing bulbous projections of the mucosa concentrated at cell margins, cobblestone structure of the surface and micropits (mP). All observations have been aided by detailed stereoscopic viewing of the micrographs. At X is a thin region where mucus granules under the surface give it a lumpy appearance. x5,200. (3) Part of (2) at higher magnification to show lateral, connecting necks between bulbous projections of the mucosa. The various stages of the process by which the "particles" could form and extrude from the mucosal surface are also demonstrated. At the arrows is evidence for supporting stalks or connecting necks being drawn up parallel to and through the surface. At the arrowhead is an example of a stalk from the surface supporting a bulbous projection, and at the clear arrow is a hole presumably left when such a stalk was torn away. x24,000. (3A) Detail from (3); atthe arrows, stalks from the surface support bulbous projections of the mucosa. x51,600. VOLUME 22, NO.3, 1976
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were outlined sharply in cobblestone relief. Unlike the normal, the numerous mucosal projections were evenly distributed over the cell surfaces, there were few if any lateral necks between them (Figure 11), and many of the projections rose with uniform thickness to considerable distances above the cell urface, resembling true microvilli, rather than bulbous projections. The tips of the projections were rounded as they were also in the normal and erosive gastritic mucosa.
DISCUSSION The epithelium of normal stomach consists of a single layer of tall, columnar, mucus-secreting cells, "the mucus surface cells". Both their ultrastructure and kinetics have been extensively studied and reported '.7 In an effort to facilitate the diagnosis of gastritis gastric biopsies have been surfaceexamined with the dissecting light microscope 8 - lo . The relatively low resolution of the light microscope has hindered progress in this effort, but interest in surface morphology of gastric mucosa in disease has revived 11-13 since the development of commercially available scanning electron microscopesl 4 with their ability to study biological surfaces directly at improved resolution and depth of field. 14 The present study has demonstrated that the tech niques of specimen preparation for scanning electron microscopy are appl icable to human stomach mucosa derived from endoscopic biopsies and that excellent preservation of ultrastruc-
tures can be achieved. The immediate fixation of the biopsy specimen without pretreatment plus the critical-point drying, as well as the higher resolution of the scanning microscope may explain why the observation of bulb-like projections of the epithelial surfaces have not been reported to our knowledge by other investigators. It is assumed that these projections are not artifactual since they are seen in some form in both normal and pathological specimens, and their shape, distribution, and frequency seem to change from one disease entity to another. Indeed, since critical-point drying is known to preserve the biological structures better than other methods of drying, artifacts could be expected to be minimal in these samples 1S . In addition, since some true microvilli are seen in all cases (the percentage being higher in gastritis) and since both bulbous projections and microvilli are seen in a single field, it is reasonable to conclude that neither structure is an artifact. It has been suggested that the lateral necks could be merely a manifestation of the metal-coating on the fibers of the dried furry coat (Figure 12). Size considerations would favor this explanation as wou Id arguments based on the geometry of the microvilli (Mv) and the furry coat in thin section. On the other hand, other observations would argue against such an explanation, particularly the great length of many of the necks and thei r observed tendency to re-enter and to re-emerge linearly from the cytoplasm (Figure 13). What seems to be a continu-
Figures: (4) Another part of (2) showing the same features as (3) and examples of thumb-like projections in the region of the clear arrows. At the solid arrows are further examples of necks apparently being pulled up parallel to and through the mucosal surface by the extruding "particles". x24,000. (5) An area of normal mucosa, showing mucus granules (e) penetrating into the lumen through an open area of the mucosa. Further examples of micropits (mP) and of stalks emerging from the surface are marked in the region of the arrowhead. xl 0,000. 140
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Figures (6) The surface of the mucosa in erosive gastritis, showing gastric pits; there is much debris with some red blood cells. x61 O. (7) Part of (6) with blebs marked at the arrows, and an area of focal erosion at the clear arrows. x4, 800. (8) Part of (7) where the bulbous projections are smaller than in the normal and are joined by necks of decreased diameter. What appears to be a long, continuous linearity joining "particle" to "particle" and emerging from and reentering the mucosal surface is marked by the arrows. x 12,000. VOLUME 22, NO.3, 1976
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Figures (9) The surface of the mucosa in chronic non-specific gastritis. Regions of focal cell degeneration are marked at the arrows. x31O. (10) Part of (9) where the cobblestone relief is very prominent. The mucosal projections are evenly distributed over the surface of the cells, and many of them are much longer and more microvillus-like than the bulbous projections described for the normal cells. x3,200. (11) Part of (1 0) where almost all of the mucosal projections appear to be thumb-like projections or true microvilli. They are distributed fairly evenly over the individual cel/surfaces, and are not joined together by necks. x 77,500. 142
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Figures: (12) A transmission electron micrograph of an isolated, heavily microvilliated area of normal human stomach mucosa, showing the microvilli in approximate cross section with the fibers of the furry coat between them. Since true microvilli as well as bulbous projections can be found in every specimen, we conclude that some components of the surface structure as seen by scanning are usually microvilli. x 36,000 (13) Normal human stomach mucosa showing micropits (mP) and, at the arrows, what appears to be a continuous rod-like neck, which connects many "particles" together, and emerges from and reenters the mucosal surface many times in the process. xIB,BOO.
ous single neck (marked at the arrows in Figure 13) enters, emerges from, and re-enters the cytoplasm, connecting a number of "particles" together as it does so. The same phenomenon is observable in mucosa in erosive gastritis (Figure 8) . Certainly, the complete nature, composition, and function of these very puzzling ultrastructures remains unexplained. In order to study the bulbous projections in cross section and at higher resolution, 2 of the specimens which had been critical-point dried, coated with gold-palladium, and examined by scanning, were re-embedded in Araldite and thin sectioned for transmission electron microscopy. It has not been possible to find fields by transmission which demonstrate the stalks and necks clearly or explain their presence, but evidence has been found in the micrographs for all of the observations reported from scanning the same sample (Figure 14). The bulbous endings were found to be membrane-bound and connected to the cell membrane by stalks and to each other by metal-coated necks. Frequently, it appeared as if a round granule (diameter 150 to 200 nm) was contained in the bulbous end. Pffeifer"'12 and others have reported the presence of microvilli in human gastric mucosa. The bulbous projections observed in addition to microvilli in the present study have the same diameter as microvilli but are somewhat shorter in length and have a different shape. The bulbous ends, supporting stalks and lateral necks of uniform caliber but VOLUME 22, NO.3, 1976
varied lengths, are not characteristics of microvilli. Nor do the lateral necks seem to be liquid or to form in any way at the expense of the bulbous bodies which they join together. Finally, the projections themselves seem to form by extrusion from the cell surface in a manner which is unlike that expected for microvilli.
Figure 14. Transmission electron micrographs of critical-point dried, gold-palladium coated, normal mucosa reembedded in Araldite after scanning, and thin sectioned. Observations of supporting stalks, joining necks, extrusion from the surface, round granules within the extremities of the bulbous projections (clear arrows), roots on some of the projections (arrow), and necks or stalks (arrowhead) being drawn up through the surface are demonstrated. x39,000. 143
Figure 15. Normal human stomach mucosa, critical-point dried and coated with goldpalladium for scanning microscopy. A region of mucosal projections and joining necks is seen to form a web over the surface. x20,OOO. Speculation about the possible function of these projections is even more hazardous than hypothesis concerning their structure. Andrew and Porter 16 have reported microvilli on the surface of mesothelial cells, proposing that they might function as a holding grid to entrap mucus and reduce friction against serosal surfaces. If the same entrapment mechanism could operate in the stomach, albeit for a different purpose, one can speculate that these projections with their interconnecting necks could eventually form a web of knotted strands to create a protective layer over the mucosal surface 17 • This could entrap secreted mucus which, after undergoing rheological changes in the new environment, could form a sort of protective membrane that contributes to mucosal resistance. That such webs do exist on the surface of normal mucosa is illustrated in Figure 15. However, complete substantiation of this would require a better understanding of both the composition and function of gastric mucus's, as well as of its interaction with cell membranes and with these projections. It is not yet possible to conclude that these projections, Iike microvilli
in the small bowel, play any part in selective absorption from the stomach. The vast increase in the numbers of true microvilli in gastritis, especially chronic, seen by both transmission and scanning electron microscopy, tends to indicate that the cells are reacting to a stimulus. It also indicates, along with their similarity in appearance, that the bulbous projections are a special form of microvilli and consequently that the microvilli and the bulbous projections form by the same extrusion process. Striking differences in surface morphology have been observed in the present study between the normal and gastritic mucosa. Because of the limited number of cases, the significance of these variations requires further investigation. Biological staining and biochemical analysis of surface structures combined with scanning and transmission electron microscopy of gastroscopic biopsies might further clarify the bioengineering design of gastric mucosa.
REFERENCES
evidence for in situ surface cell degeneration. Exp Mol Patho/13:19, 1970 12. PFEIFFER CI. GEOFFREY R, JOSEF W: Gastrointestinal ultrastructure, in, An Atlas of Scanning and Transmission Electron Micrographs, Academic Press, New York and London, 1974 13. KATSU K: Scanni ng electron microscopic study of the gastroi ntesti naltract; 3-dimensional observation of the mucosal surface. lap I Gastroengerology (in japanese) 70:812, 1973 14. WATSON JHL: The scanning electron microscope. Henry Ford HospMedl 21:75,1973 15. POLLIACK A, LAMPEN N, deHARVEN E: Comparison of air drying and critical point drying procedures for the study of human blood cells by scanning electron microscopy. In: IITRIISEM.Ed. johari and Cowin, Chicago, 1973 16. ANDREWS PM, PORTER KR: The ultrastructural morphology and possible functional significance of mesothelial microvilli. Anal Rec 177:409, 1973 17. WATSON JHL, GOODWIN, J, FALLAH E: Some observations on the ultrastructure of the surface of normal human stomach mucosa. In 33rd Ann Proc Electron Microscopy Soc Amer. Ed. GW Bailey. Claitor's Publishing Division, Baton Rouge, 1975 18. SCHRAGER J: The chemical composition and function of gastrointestinal mucus. Gut 11 :450, 1970
1. PALMER ED: Gastritis: a revaluation. Medicine 33:199, 1954 2. WOOD II. TAFT 1I: Diffuse Lesions of the Stomach. Edward Arnold, london, 1958 3. DAVENPORT HW: Back diffusion of acid through the gastric mucosa and its physiologic consequences. In, Progress in Gastroenterology, Vol. 2, GBj Glass, Ed. London, Grune and Stratton, Inc., P. 42, 1970 4. ANDERSON TF : Techniques for the preservation of 3-dimensional structure in preparing specimens for the electron microscope. Tr N Y Acad Sci 13:130, 1951 5. WATSON JHL: Unpublished research, 1974 6. LILLIBRIDGE CB: The fine structure of normal human gastric mucosa. Gastroenterology 47:269, 1964 7. RUBIN W, Ross LL, SLEISENGER MH, JEFFRIES GH: The normal human gastric epithelia. Lab Inv 19:98, 1968 8. BURHOL PG: Surface microscopic and histologic appearances of the gastric mucosa. Scand I Gaslroenterol 3: 17, 1968 9. SALEM SN, TRUELOVE SC: Dissecting microscope appearances of the gastric mucosa. Br Med I 2: 1S03, 1964 10. SALEM SN: Gastric mucosa under the dissecting microscope. Am I Dig Dis 10:705, 1965 11. PFEIFFER CJ: Gastric surface morphology in man, monkey and ferret;
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