Gluten-sensitive enteropathy: Sequence of villous regrowth as viewed by scanning electron microscopy

GLUTEN-SENSITIVE ENTEROPATHY: Sequence of Villous Regrowth as Viewed by Scanning Electron Microscopy Susan A. Halte~, MD,* Harry Lee Greene, MD, t and Gerard Helinek, MD'? Small-intestlnal biopsy specimens from five c h i l d r e n with gluten-sensltlve enteropathy (GSE) were studied by scanning electron microscopy (SEM) before dietary treatment and sequentially after removal of gluten from the diet. Compared with biopsy specimens from patients without evidence of villous atrophy, these specimens showed marked architectural changes. In patients with total villous atrophy observed by light microscopy, absent villi and prominent crypts were observed by SEM. Patients with subtotal villous atrophy had the first stages of villous formation characterized by semicircular ridge formation. Biopsy specimens obtained from the five patients with GSE after dietary treatment demonstrated a sequence of changes characterizing villous repair. The earliest change consisted of semicircular elevations of cells around the crypts. Unequal cellular repletion around the crypts and migration of the cells led to the formation of parallel ridges. Increased cell proliferation resulted in thickening and twisting of the ridges to form convoluted ridges. Increased complexity of the ridges appears to lead to the formation of cerebriform ridges, and septatlon at various points in the ridges results in the formation of mltten-like villi. In biopsy specimens from patients with villous atrophy from other causes, similar reparative changes were seen by SEM. Scanning electron microscopy is a useful adjunct to the light microscopic and dissecting microscopic assessment of small intestine biopsy specimens, and may be more sensitive than light microscopy in documenting early morphologic response to dietary treatment for gluten.sensitive enteropathy. Hum Pathol 13:811--818, 1982.

The histologic features of gluten-sensitive enteropathy (GSE) have been well described, as has the sequence of villous regrowth following dietary restriction o f glttten. ~-3 However, a three-dimensional view of the sequence o f intestinal repair has not been fully appreciated. Since scanning electron microscopy (SEM) allows three-dimensional viewing o f villous growth, this study was undertaken to document the various stages o f villous regrowth in patients with GSE after elimination o f dietary gluten, as well as to determine whether the small-intestinal mucosa actually returns to normal with gluten restriction. In addition, a few observations in patients with other diseases known to cause villous damage suggest that villous repair from an insult proceeds through a similar series of changes during healing. MATERIALS AND METHODS

Small-intestinal biopsy specimens were obtained with a pediatric 1.8-mm double-port Crosby-Kugler Received from the Departments of Pathology* and Pediatrics,t Vanderbilt University School of Medicine, Nashville, T N 37232. Accepted for publication January 6, 1982. Address correspondence and reprint requests to Dr. Halter.

biopsy capsule. 4's Tile capsule was localized by fluoroscopy just distal to the ligament o f Treitz before tile tissue was obtained. The specimens were examined and oriented on lens paper with ttle aid of a dissecting microscope. T h e portion to be examined by light microscopy was fixed in 10 per cent buffered formalin, dehydrated, and embedded in paraffin in a standard manner. T h e specimen was left attached to the lens paper to facilitate proper alignment during embedding in paraffin. Sections 5/sm thick were cut and stained with hematoxylin-eosin. Specimens prepared for SEM were fixed for 24 to 72 hours in 3 per cent glutaraldehyde in 0.1-molar cacodylate buffer at pH 7.4. Tissues were washed once with distilled water and postfixed for two hours at room temperature in 1 per cent osmium tetroxide in 0.1-molar cacodylate buffet" at p H 7.4 and rewashed with distilled water. The tissues were dehydrated through gradually increasing concentrations of ethanol and critical-point dried in a Bomar SPC1500 critical-point drier using liquid carbon dioxide. Specimens were mounted on aluminum stubs, and excess mucus was removed mechanically using a fine wire brush and forcing compressed air through a 26gauge needle. The specimens were coated with 10 nm of gold-palladium in a H u m m e r II apparatus and viewed at 20 kV in Hitachi S-500 scanning electron microscope. Two groups of patients were evaluated: 1) five children with GSE, and 2) 18 patients, ranging in age from 2 months to 48 years, with chronic diarrhea and normal histologic features of the jejunal mucosa observed by light microscopy. The latter patients served as controls, since fecal fat excretion and one-hour D-xylose absorption were normal. Either two or three biopsy specimens from patients with GSE were examined by light microscopy and SEM. A total of 14 biopsy specimens from children with GSE were examined (table 1). All but two patients were biopsied within 12 hours o f the last gluten-containing meal. Specimens were then obtained at various intervals after dietary restriction of gluten, to study the progression of mucosal regrowth. At the time o f initial biopsy, two children (patients 1 and 2) had total villous atrophy or a completely flat mucosal pattern observed by light microscopy. The biopsy specimen from one patient (patient 5) showed subtotal villous atrophy. The two remaining patients (patients 3 and 4) had been admitted with severe diarrhea, malnutrition, and dehydration and required parenteral nutrition. Patient 4 received par-

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TABLE 1, C L I N I C A L DATA AND H I S T O L O G I C A P P E A R A N C E OF J E J U N A L MUCOSA OF P A T I E N T S W I T H G L U T E N - S E N S I T I V E E N T E R O P A T H Y Age (years) Patient 1

1.2

Patient 2

5

Patient 3

1.8

Patient 4

2

l'atient 5

14

Initial

Initial

Biopsy

SEM

Total villous atrophy Total villous atrophy Subtotal villous atrophy Total villous atrophy Subtotal villous atrophy

Absent Villi

Rebiopsy Date

Clinical Status At Rebiopsy

Rebiopsy LM

Rebiopsy SEM

4 mo

Clinical improvement, weight gain

Total villous atrophy

Circular ridges

5 mo

Clinical improvement, weight gain Clinical improvement, weight gain

Total villous atrophy Subtotal villous atrophy Subtotal villous atrophy Normal villous crypt ratio Subtotal villous atrophy Subtotal villous atrophy

Semicircular ridges Convoluted ridges

Absent Villi

4 wk

SemiCircular ridges Circular ridges

4 mo

Clinical improvement, weight gain

6 wk

Clinical improvement, weight gain

Convoluted ridges

1 wk

Little clinical improvement, no weight gain Clinical improvemeut

1 yr

enteral nutrition for two days before biopsy, and patient 3 was treated for five days before initial biopsy. Since the initial appearance of the inucosa conld not be determined in these two patients, their biopsy specimens actually showed the clmnges observable after two and five days, respectively, of a glutenfree diet. A second small-intestinal biopsy was performed on each patient--after one week (patient 5), four weeks (patient 2), six weeks (patient 4), four lnonths (patients 1 and 3), and five months (patient 1) o f dietary restriction of ghtten. All patients except patient 5 showed clinical improvement with weight gain after dietary t r e a t m e n t (table 1). After two additional weeks of gluten restriction, patient 5 began to show clinical response to the diet. Patient 1 had poor compliance with gluten restriction, and in spite of clinical improvement after treatment for four months, the second biopsy specimen continued to show total villous atrophy by light microscopy. Because of doubt concerning the diagnosis, a third biopsy was performed on this patient one month later. On each patient with chronic diarrhea and normal smail-intestilml histologic features, only a single biopsy was performed. Since no architectural abnormalities were observed by light inicroscopy in this g r o u p o f 18 biopsy specimens, they served as a baseline (control group) against which the abnormal specimens were judged. RESULTS

CONTROL PATIENTS. Small-intestinal biopsy specimens from patients with chronic diarrhea and normal growth curves had epithelium that appeared normal by light microscopy (fig. 1). Scanning electron microscopy showed prominent villi with three main

812

Cerebriform ridges Convoluted ridges Cerebriform ridges Cerebriform ridges

configurations (fig. 2): Finger-like villi were tall, with the diameter of the tip approximately equal to that of the base (fig. 2, top left). Other villi had a base that was greater in diameter than the tip, giving them a leaflike configuration (fig. 2, top right). The third type of villous configuration consisted of a broad base and tip, making these villi look like mittens (fig. 2, bottom left). One or several types of villous configttratiou could be seen in a single specimen. Many villi were traversed by fttrrow-like depressions. Crypts were not ahvays exposed, but in specimens that were focally stretched, they could be seen as single orifices in the mucosal floor (fig. 2, bottom right). Only rarely were vestibules with mntiple openings seen. The epithelial cells of the villi were regular in slmpe. In some villi, focal mucous production was suggested, identifying a mucus-secreting or goblet cell. Microvilli could not be seen because of the thick glycocalyx that obscured the surface of the epithelial cells. PATIENTS WITtt GLUTEN-SENSITIVE ENTEROPA-

Wily. The initial biopsy specimens from three patients (patients 1, 2, and 4) showed total villous atrophy when viewed by light microscopy. T h e crypts were elongated and contained many mitotic figures. The lamina propria was infiltrated by numerous lymphocytes, plasma cells, and eosinophils (fig. 3, top). The SEM appearance of the initial biopsy specimens of these patients was not identical. Two of the three specimens with total villous atrophy (patients 1 and 2) were completely flat when viewed by SEM (fig. 3, bottom left). No villi were present, and crypt openings were large and prominent. The biopsy specimen from patient 4, however, showed circular elevations or ridges of cells around the crypt openings (fig. 3, bottom right). Since this patient had received parenteral nutrients and no dietary gluten for 48 hours prior to the biopsy, these changes may have repre-

GLUTEN-SENSITIVE ENTEROPATHY ON SE~I--HALTER ET AL.

Figure 1. Lightmicrograph of histologicallynormal, well-oriented biopsyspecimen from control. The villous crypt ratio is normal. (Hematoxylin-eosinstain. x156.)

sented tile earliest stage in the reparative process. As viewed by SEM, the epithelial cells on the mncosal surface o f all three patients were irregular in size and shape and crypts were prominent. In the initial biopsy specimen of two patients (patients 3 and 5), subtotal villous atrophy was apparent on light microscopy (fig. 4, top). In these specimens the crypts were elongated and there were numerous mononuclear cells in the lamina propria. Villi were present but appeared bltmted. There was great variation in the SEM appearance of the two cases. The biopsy specimen from patient 3, who had received parenteral feedings without gluten for five days before initial biopsy, showed ridges o f cells around the crypts (fig. 4, bottom left), which were clearly higher than those in patient 4, who had received parenteral feedings for only two days. In addition, each side of the ridge had a cleft formation (fig. 4, bottom left, arrows). This gave the appearance o f two semicircles of cells accumulating around the crypt openings, and suggested the second stage of mucosal repair. The SEM appearance o f the initial biopsy specimen in patient 5 suggested a further stage o f the reparative process. Cells from one ridge along the crypts appeared to spread to meet the cells from the adjacent ridge (fig. 4, bottom right). Biopsies were p e r f o r m e d on all patients with GSE after they had been on gluten-free diets for vary!ng periods o f time. In all cases, there was progression m the reparative process, although the degree o f improvement did not always correspond to the length o f time on the diet. For example, patient 1 was rebiopsied five months after starting treatment and had only attained the stage of semicircular ridges (fig. 5, top /eft), whereas patient 4 was rebiopsied at six weeks and had progressed to a stage where the cellular ridges had become twisted or convoluted (fig. 5, top right). With continued proliferation and migration o f cells, the pattern o f the ridges became more complex. Patient 2 was rebiopsied at four weeks; by SEM, the

ridges were seen to have become thick and twisted, imparting a more complex, convoluted pattern to tile mucosa (fig. 5, bottom left). By light microscopy, the rebiopsy specimen from this patient showed subtotal villous atrophy. Scanning electron microscopy of the biopsy specimen taken from patient 3 four months after dietary treatment demonstrated a final step in the repair process. Although on light microscopy the biopsy specimen obtained from this patient at four months showed only slight improvement over the initial specimen, on SEM it showed tile formation of cerebriform ridges with focal areas suggestive o f septation into villi (fig. 5, bottom right). In areas o f this biopsy specimen, large mitten-like villi were also present. This final stage o f regrowth wouhl be expected eventually to result in a normal mucosal pattern. A h h o u g h different stages o f mucosal repair could be determined by SEM, none of the patients showed complete return to a normal mucosa. Rebiopsy o f patient 5 performed after two years on a gluten-restricted diet did not reveal a completely normal pattern. Ahhough these findings may represent failure of dietary compliance, they also could indicate that patients with GSE may never have complete recovery o f a normal pattern, as viewed by SEM. DISCUSSION

Gluten-sensitive enteropathy, also known as celiac disease and nontropical sprue, is a malabsorptive disorder characterized morphologically by villous atrophy, crypt hyperplasia, and marked infiltration of the lamina propria with plasma cells, lymphocytes, and eosinophils. 6-s The disorder occurs in a small number of persons who are sensitive to a protein complex called gluten, which is found in a variety of cereal grains. Ahhough the pathogenesis of this disease in susceptible individuals is unknown, either an abnormal immunologic response or an unidentified inborn metabolic defect has been postulated as its

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Figure 2. Composite scanning electron micrographs of biopsy specimens from controls. Top left, finger-like villi (arrow) from the same specimen depicted in figure 1. (x47.) Top right, leaf-like villi (arrow) are broader at the base than at the tip. (x44.) Bottom left, mitten-like villi (arrow) have broad bases and tips. (x63.)Bottom right, furrow-like depressions are present on the villi (open arrow head). Crypts are single openings (short arrow) or vestibules with multiple openings (long arrow). (x230.)

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GLUTEN-SENSITIVE ENTEROPATHY ON SEM--HAL'rER ET AL.

Figure 3. Total villous atrophy. Top, light micrograph of biopsy specimen from patient with GSE (patient 4) shows absence of villi, increased crypt depth, and numerous chronic inflammatory cells in lamina propria. (Hematoxylin-eosin stain. • Bottom left, scanning electron micrograph of biopsy specimen from patient I shows prominent crypt openings and absent villi. (• right, scanning electron micrograph of biopsy specimen from patient 4 shows slight elevations of the epithelial cells around the crypts (arrow). (• cause. 9 Since elimination o f gluten from the diet resuits in a gradual restoration o f the small-intestinal epithelium to normal, GSE provides a u n i q u e opportnnity to study villous renewal. Biopsy specimens were obtained f r o m 18 patients in whom histologic features o f the small intestine app e a r e d n o r m a l on light microscopy; these were used for c o m p a r i s o n with biopsy specimens f r o m the patients with GSE. T h e specimens f r o m the 18 Control patients showed villous projections that a p p e a r e d either finger-like, leaf-like, o r mitten-like o n SEM, identical to those seen in biopsy specimens f r o m normal adults. 1~ In this r e p o r t several stages o f epithelial renewal

after dietary t r e a t m e n t for GSE were d e m o n s t r a t e d by SEM. O n the basis o f assessment o f the a p p e a r a n c e o f the jejunal mucosa o f five patients b e f o r e and after the start o f a gluten-free diet, the following schema for epithelial repair o f the h u m a n jejunal mucosa has b e e n s u g g e s t e d : C o m p l e t e a b s e n c e o f villi with p r o m i n e n t crypt openings is seen as the most severe stage o f the disease. A f t e r gluten is r e m o v e d f r o m the diet, cells migrating out o f the crypts accumulate in a circular ridge a r o u n d the crypts. This initial oval ridge o f cells eventually forms bilateral clefts or a semicircular c o n f i g u r a t i o n as m o r e cells accumulate in an u n e v e n m a n n e r . Parallel ridges are f o r m e d when the arms o f the oval ridges elongate. T h e s e

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Li

| B

Figure 4. Subtotalvillous atrophy. Top, light micrograph of biopsy specimen from patient 3. Crypts are elongated and villi are short and irregular. (Hematoxylin-eosin stain, x424.) Bottom left, biopsy specimen from patient 3 has prominent crypts surrounded by opposing ridges of cells that have clefts (arrows) at opposite ends, suggesting a semicircle configuration. (• Bottom right, light microscopic appearance of specimen from patient 5 is similar to that shown in figure 4, top (patient 3). Mucosa is composed of parallel ridges joined by projections of cells. (x447.)

c o n f l u e n t extensions f o r m ridges between crypts. As m o r e cells accumulate, the ridges b e c o m e m o r e confluent to f o r m long convolutions. Finally, in areas w h e r e cells accumulate most rapidly, mitten-like villi are p r o d u c e d by septation o r f u r r o w i n g o f the convolutions. Marsh t4 followed two patients with GSE by SEM after six weeks and 12 m o n t h s o f dietary t r e a t m e n t , respectively. A l t h o u g h the first patient achieved a n o r m a l - a p p e a r i n g mucosal pattern by nine months, the mucosa o f the second patient had r e a c h e d only the convoluted-ridges stage two years a f t e r dietary treatment. In contrast, the jejunal mucosa o f patient 5

816

in tile present series had evidence o f c e r e b r i f o r m ridges by six weeks o f dietary treatment. T h e lack o f correlation between clinical symptoms and histologic a p p e a r a n c e o f the jejunal biopsy is well known, in T h i s was also observed in this study, but the SEM a p p e a r a n c e correlated m o r e closely to the clinical picture in m o r e patients than did the appearance on light microscopy. Although a patient's clinical response is p e r h a p s the most reliable m e t h o d o f assessing overall response to treatment, SEM may be m o r e sensitive than light microscopy in d o c u m e n t i n g early m o r p h o l o g i c response. Scanning electron microscopy is also able to

GLUTEN-SENSITIVE ENTEROPATHY

O N SEM--HALTER ET AL.

Figure 5. Scanning electron micrographs after treatment with gluten-free diet. Top left, five months after dietary treatment, mucosa of patient 1 has a semi-circular configuration. (• Top right, six weeks after dietary treatment, mucosa of patient 4 is composed of convoluted ridges. Surface is partially obscured by mucus. (x265). Bottom left, four weeks after treatment, mucosa of patient 2 demonstrates more complex convoluted ridges. (x200.) Bottom right, four months after treatment, mucosa of patient 3 has cerebriform ridges with evidence of septation into villi (arrow). (x200.)

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demonstrate the stepwise progression o f the epithelial repair process m o r e precisely titan can light microscopy. Light microscopy assessment o f small-intestinal biopsy is specimens is limited by orientation. Diagnosis is o f t e n restricted to b r o a d categories such as "subtotal," "total," o r "severe" villous a t r o p h y . By SEM several subcategories u n d e r the light microscopic description o f total villous a t r o p h y have been d o c u m e n t e d , including circular ridges a r o u n d the crypts, elevation o f the ridges, and u n e q u a l migration o f the cells to f o r m clefts on the initially circular ridges. Scanning electron microscopy has revealed that subtotal villous a t r o p h y also consists o f several stages. Semicircular ridges as well as convolution o f the ridges can be seen at this stage. Subtotal villous a t r o p h y may also include c o m p l e x convolutions o f the ridges and the d e v e l o p m e n t o f septations o r furrows resulting in thick mitten-like villi. T h e use o f the dissecting microscope in the assessment o f small-intestinal biopsy specimens b e f o r e fixation has been c o n s i d e r e d by H o h n e s et al. aG to be helpful in the diagnosis and follow-up o f patients, but by Perera et al. t5 to be o f little value. A l t h o u g h the flat mucosal a p p e a r a n c e in severe GSE may be readily d o c u m e n t e d , the intermediate stages are less easily seen, for a variety o f reasons, including the presence o f m u c u s a n d the lack o f contrast in the tissue. It is possible to note man), m o r e changes by SEM than is possible with the dissecting microscope, which suggests that SEM is a better tool for studying the seq u e n c e o f villous r e p a i r . A l t h o u g h p r o c e s s i n g for SEM takes m o r e time t h a n i m m e d i a t e o b s e r v a t i o n u n d e r the dissecting microscope, specimens can be processed a n d viewed within 24 hours. In addition, SEM may be a useful adjunct to the light microscopic and dissecting microscopic assessm e n t o f patients with o t h e r diseases that cause villous a b n o r m a l i t i e s . A l t h o u g h villous r e g e n e r a t i o n was e x a m i n e d in detail only in patients with celiac disease, in two patients with intractable d i a r r h e a a n d nialnu-

818

trition who were e x a m i n e d sequentially, villous regrowth a p p e a r e d to follow the same sequence.

REFERENCES 1. Yardley JH, Bayless TbI, Norton JH, et al: Celiac disease: a study of the jejunal epithelium before and after a glutenfree diet. N EnglJ bled 267:1173, 1962 2. Anderson CbI: Histologicalchanges in the duodenal mucosa in coeliac disease: reversibility during treatment with a wheat gluten free diet. Arch Dis Child 35:419, 1960 3. Young WF, Pringle Ebl: 110 children with coeliac disease, 1950-1969. Arch Dis Child 46:421, 1971 4. Crosby WH, Kugler HW: lntraluminal biopsy of the small intestine. Am J Dig Dis 2:236, 1957 5. Greene HL, Rosensweig NS, Lufkin EG, et al: Biopsy of the small intestine with the Crosby-Kugler capsule: experience in 3,866 peroral biopsies in children and adnhs. Am J Dig Dis 19:189, 1974 6. Dicke WK, Weijers HA, Kamer VD: Coeliac disease. II. The presence in wheat of a factor having a deleterious effect in cases of coeliac disease. Acta Paediatr 42:34, 1953 7. Van de Kamer H, Weijers HA: Coeliac disease. V. Some experiments on the cause of the harinful effects of wheat gliadin. Acta Paediatr 44:465, 1955 8. Katz AJ, Falchuk Zbl: Current concepts in gluten sensitive enteropathy (celiac sprue). Pediatr Clin North Am 22:767, 1975 9. Falchuk Zbl: Update on gluten-sensitive enteropathy. Am J bled 67:1085, 1979 10. Demling L, Becker V, Classen hi: Examinations of the mucosa of the small intestine with the scanning electron microscope. Digestion 2:51, 1969 11. Asquith P, Johnson AG, Cooke WT: Scanning electron microscopy of normal and celiacjejunal mucosa. Am J Dig Dis 15:511, 1970 12. Toner PG, Cart KE: The use of scanning electron microscopy in the study of the intestinal villi. J Path 97:61 l, 1969 13. blarsh biN, Swift JA: A study of the small intestinal mucosa using the scanning electron microscope. Gut 10:940, 1969 14. blarsh Nbl: The scanning electron microscope and its application to the investigation of intestinal structure, ht Badenoch J, Brooke BN (eds): Recent Advances in Gastroenterology. Baltimore, Williams & Wilkins, 1972, p 81 15. Perera DR, Weinstein WM, Rubin CM: Small intestinal biopsy. Hum Pathol 6:157, 1975 16. Holmes R, Hourihane DO'b, Booth CC: The mucosa of the small intestine. Postgrad bled J 37:717, 1961