CROHN'S DISEASE: A SCANNING ELECTRON MICROSCOPIC S T U D Y * Ann M. Dvorak, M.D., t Anne B. Comtell, B.A.,-~ and G. Richard Dickersin, M.D.w



A scanning electron microscopic study of Crohn's disease was done using surgically resected specimens. Grossly normal resection margins as well as nonulcerated portions from diseased areas were selected for study. Scanning electron microscopic findings in Crohn's disease included changes in villous size and shape, villous fusion and epithelial bridge formation, goblet cell hypertrophy and hyperplasia, and increased secretion of mucus. These changes were marked in involved areas, and many were also present in six of seven margins of resection available for study in the ileal group. The abnormalities found in grossly normal margins of resection suggest a more widespread involvement than can be appreciated by gross and light mic~igscopic examination of the specimen. Formation of the increased coat of mucus observed may be stimulated by a number of agents and could contribute to an enhanced barrier function in areas of earl}' involvement in Crohn's disease, thereby decreasing the uptake of toxic or antigenic macromolecules. A decrease in bacterial sttperinfections would also be facilitated. Later lesions, with severe villous changes and hypersecretion of mucus, may favor the uptake of toxic and antigenic macromolecules as well as aid in the establishment of bacterial superinfections.

Crohn's disease was first described in 1932 and remains a disease of unknown etiology? Patients are variously symptomatic with crampy abdominal pain, diarrhea, constipation, partial bowel obstruc-

tion, bowel perforation, and formation of fistulas. Tile disease is characterized by recurrences following surgical resection and may be suppressed but not cured by medical therapy. Witll the advent of im-

*This work was supported in p~irt by grants CA 19141 from the National Cancer Institute and IM 44A from the American Cancer Society. tAssociate Professor of Pathology,Harvard MedicalSchool. Associate Pathologist,Massachusetts General Hospital,Boston,Massachusetts. :[:SeniorTechnician,MassachusettsGeneral Hospital,Boston,Massachusetts. w Professor of PathologT, Harvard MedicalSchool. Associate Pathologist, Massachusetts General Hospital,Boston,Massachusetts.


HUMAN PATHOLOGY--VOLUME 10, NUMBER 2 March 1979 proved techniques for viewing uncoated biologic specimens by scanning electron microscopy, we used these techniques to study nonulcerated involved areas of surgical specimens from patients with Crohn's disease, as well as to study the resp.ective grossly normal margins of resection. MATERIALS A N D M E T H O D S

Surgical specimens from 12 patients with Crohn's disease and one patient with carcinoma of the colon provided material for this study. The diagnosis of Crohn's disease was confirmed using standard clinical, radiographic, and pathologic criteria3 ,3 Nonulcerated portions of diseased areas and portions of grossly normal appearing resection margins were selected and fixed by immersion for five hours at room temperature in a mixture containing 2 per cent paraformaldehyde, 2.5 per cent glutaraldel]yde, and 25 rag. of calcium chloride in 0.1 M sodium cacodylate buffer (pH 7.4). 4 Samples were further processed using Malick and Wilson's modification of the thiocarbohydrazide mediated osmium binding technique, dehydrated in graded alcohols, critical point dried using carbon dioxide, and viewed in an AMR 1000 scanning electron microscope? -~ RESULTS


Previous descriptions of human small gut mucosa have included a variety of specimen preparation techniques, all of which employed evaporated heavy metal layers and primarily freeze drying for removal of water from tissnes, s-n One study used critical point drying and Malick and Wilson's modified thiocarbohydrazide mediated osmium binding method, but these authors sputter coated a 100 /~ layer of surface gold as well. '2 In an attempt to see the tissues as nearly in their normal state as is possible with present day technology, we purposefully did not wash the samples prior to fixation (to enable us to accurately study mucus secretion and layers), and after using Malick and Wilson's modified technique and

critical point drying we did not evaporate or sputter any heav.y metal surface layers prior to examination in the scanning electron microscope. The samples so prepared presented no difficnlties with charging effects and were stable for long viewing times as well as long storage times for re-examination. The normal ileal mucosa had clearly delineated villi, primarily o f two shap'esfinger shaped and leaf or tongne shaped (Fig. 1). Cross sections of these structures had an oval shape, and the external epithelial surface as well as the fibrovascular lanfina propria core conld be seen. IndMdual goblet cells filled with granules of mucus conld be easily identiffed, and specimen tilting in the electron microscope permitted positive correlation of these underlying granules of nmcus with the normal small orifices of goblet cells on the villous surfaces. Normal ileal villi without heavy metal surface layers showed characteristic definition of the surface. Villous folds were present, and these were predominantly located horizontally. Some were also oriented verticall2, in-relationship to the long axis of theft, ill{. The hexagonal outline of indMdu,'ll.epithelial cells was easily visible at magnifications as low as 350• Infrequent small pits corresponding to orifices of goblet cells were present. These orifices were usually devoid of mncns and did not show distention or cracking at their edges. Occasional secretion of mucus could be seen originating from some such orifices. Tips of villi of these umvashed samples were usually devoid of attached mucus and debris. Occasionally extrusion of a dead epithelial cell from the villous tip was present. The Inicrovilli of the epithelial cells were visible at a magnification of 5000• and they were clearly visible at 10,000x. Scanning Electron Microscopy in Crohn's Disease Limited to the Ileum

DISEASED ILEUM. Some specimens retained visible villous structures that invariably showed alterations in size a n d shape (Figs. 2 to 4). Marked increases in the thickness of villi, shortened villi, and irregularly shaped villi were noted. Fu-






Figure 1. Nor,hal ileum as seen by scanning electron microscopy from a 70 ye~.|r ok1 male. A low magnification survey picture (d) o f the tissue shows n u m e r o v s leaf and finger shaped villi o f nornml sizes a n d shapes. A few a d h e r e n t strands o f ,nucus are present. In B a higher magnification shows minimal goblet cell orifices and no a d h e r e n t mnctls. Villi are well delineated. C, D, and E show Icaf :and finger villi, respectively, at sufficient magnification to see the minute goblet cell orifices (arrows) o f the normal ileum. Mucus extrusion is p r e s e n t from several o f these in D (dotted arrows). 1lorizontal and longitudinal villous folds are seen best in C (open arrows). In E the hexagonal outline o f individual epithelial cells is easily seen (broken arrows). T h e s e are m o r e clearly seen in F s u r r o u n d i n g the orifice o f a goblet cell (arrow). (,I, x 23. B, • 75. C, x 21'1(}.D, x 175. E, • q00. F, X 3000.)

(Text continued on page 171)




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Figure 2. This ileal sample was obtained fiom a 77 year okl male with a two day history of symptoms, perforation, and no d~erapy prior to surgery. T h e survey lfi~ture (A) at low magnification shows broad, thick, and fiat villi with n u m e r o u s tixsioq points (arrows). In B the thick, club s h a p e d villi show fusion (broken arrows) and large n u m b e r s o f enlarged goblet cell orifices (arrows). C slmws a small aphthous ulcer (large arrow) surr o u n d e d b)" a concentric ociemation o f blunt villi with fusion (small arrow) and large n u m b e r s o f goblet cell orifices. D shows the blunt tip o f one villus with mxmecous goblet cell orifices, whereas E and F show a side view o f thickexmd, b h m t villi with increases in goblet cells9 In (;, the vast pavement o f the surface by distemled, mucus secreting goblct cells (arrow) in Cr.ohn's disease is evident. (A, • 23. B, • 100. C, • 100. D, x 200. E, x I 15. F, x.88. G, x210.)



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Figure 3. llet,m from an 11 year old male with a one year histor)' o f s y m p t o m s and no prior d r u g t h e r a p ) shows thick, broad, and lilt villi i,l the diseased portion (,-1, C, F) with a marked increase ill goblet cells. T h e resection margin (B) had villous size and shape changes, as well as ah increase ill goblet cells. D :rod E are higher magnification views to show the mucigen granules filling the distended goblet cell orifices. Some granules are free on the epithelial cell surface (arrow). Interepithelial cell and goblet cell cracks are seen in E (arrows). T h e inset in D shows a large globule n f mucus in the process o f extrusion from all orifice, and the inset in E is o f a fractured fissile plane, which shows the u n e x t r u d e d mucigen granules o f two goblet cells beneath the cell surface (arrows). (A, • B, • C, • D, • inset, • 1000. E, • inset, • F, •



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Crohn's disease: a scanning electron microscopic study.

CROHN'S DISEASE: A SCANNING ELECTRON MICROSCOPIC S T U D Y * Ann M. Dvorak, M.D., t Anne B. Comtell, B.A.,-~ and G. Richard Dickersin, M.D.w Abstract...
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