Digital Morphometric and Stereologic Analysis of Small Intestinal Mucosa in Well-Nourished and Malnourished Children with Persistent Diarrhea

ORIGINAL ARTICLE

Digital morphometric and stereologic analysis of small intestinal mucosa in well-nourished and malnourished children with persistent diarrhea

Ana Luiza G. Pires,1 Themis Reverbel da Silveira,2 Vinícius Duval da Silva3

Abstract Objective: to test the hypothesis that the proximal small intestines of children with persistent diarrhea present morphometric and stereologic changes proportional to their nutritional status, using microscope images stored in a computer. Methods: cross-sectional study with 65 pediatric patients, whose ages ranged from 4 months to 5 years, with persistent diarrhea for over 14 days. The nutritional assessment was performed according to the z- scores for weight/age (W/A), weight/height (W/H) and height/age (H/A) ratios, divided into: well- nourished = z > 2SD and malnourished = z < -2SD; well-nourished = z > 2SD, nutritional risk = z < -1SD and malnourished = z < -2SD; and continuously, in descending order, using the NCHS charts. After obtaining the computer images using the software Scion Image, villous height, crypt depth, mucosal thickness, total mucosal thickness, and villous/crypt ratio were measured in the fragments of the small intestinal mucosa, enlarged 100 times. When images were enlarged 500 times, enterocyte height, nuclear height and brush-border height were measured. Stereologic analysis was performed using cycloid arcs. Results: for W/A, W/H and H/A z-scores, divided into two nutritional status categories, no statistically significant difference was observed in regard to villous height, crypt depth, mucosal thickness, total mucosal thickness and villous/crypt ratio. Enterocyte height presented the most significant difference between well-nourished and malnourished groups, for W/A and W/H ratios, with a 500x enlargement, although this difference was not statistically significant. When z-scores were subdivided into three nutritional status categories, a digital morphometric analysis showed a statistically significant difference for villous/crypt ratio between the well-nourished and slightly malnourished group and the well-nourished and mild to severe malnourished group (p = 0.048). The villous/crypt ratio was higher among well- nourished children. Using the Spearman coefficient, the variables enterocyte height, height of enterocyte nucleus and brush-border height presented a clear association with the W/A ratio (r = 0.25; p = 0.038), W/ H ratio (r = 0.029; p = 0.019). The height of the enterocyte and the brush-border height were associated with W/H ratio. Conclusions: the observed associations between nutritional status and the analyzed small intestinal mucosa variables showed a positive correlation with patients’ weight. Although these associations were of a slight to moderate magnitude, we observed a tendency of enterocyte size reduction, as well as a reduction in the size of its nucleus and brush-border, as the level of malnutrition increases.

J Pediatr (Rio J) 2003;79(4):329-36: diarrhea, malnutrition, digital morphometric analysis, stereologic analysis.

1. PhD. Specialist in Pediatric Gastroenterology, Hospital Materno Infantil Presidente Vargas. 2. Associate Professor, Universidade Federal do Rio Grande do Sul. 3. PhD in Pathology, Universidade Federal do Rio Grande do Sul. Manuscript received Oct 15 2002, accepted for publication May 22 2003.

329 330 Jornal de Pediatria - Vol. 79, Nº4, 2003 Digital morphometric and stereologic analysis... – Pires ALG et alii

Introduction technique of counting cycloid curves superimposed upon According to the World Health Organization, severe the images was used. malnutrition is a social and medical problem and is associated The current study used an easily accessed program with 29% of child deaths in the 0 to 4 years age group.1 which is available on the Internet (Scion Image Rasband Pelletier et al.,2 however, believe that this percentage is not W. Scion Image Beta2 on line version),17 in order to a true reflection of reality and consider that 56% of child evaluate, by means of computerized morphometry, deaths are due to damage resulting from milder forms of alterations caused by malnutrition to the small intestine malnutrition. The pathogenesis of persistent diarrhea mucosa of well-nourished children and of children with associated with malnutrition is multifactorial, with early differing degrees of malnutrition. weaning, poverty, infectious and nutritional factors and allergic phenomena contribute to its perpetuation. The final result of these events is damage to the small intestine Methods mucosa. An understanding of the complex interactions The histological material was obtained from stored which take place in the luminal-mucosal interface of the blocks of wax from 85 hospitalized children, the majority small intestine is fundamental to understanding the nature at the Pediatric Gastroenterology Unit at the Santa Casa of this type of diarrhea.3 de Misericórdia (Blessed House of Mercy) in Porto Clinical and experimental studies of humans have shown Alegre, in the period between May 1989 and November that nutritional disturbances can produce morphological 1991. For the current study children were selected aged 4,5 and structural alterations to the intestinal mucosa. These between 6 months and 5 years, with diarrhea of more than alterations can be reflected in morphometry and in the 14 days’ duration who had had a small intestine biopsy as surface/volume ratio of the intestinal mucosa. part of the work up for to investigate the diarrhea. In order to study these alterations, sensitive, refined Nutritional status was assessed by means of z scores for techniques which can be easily reproduced must be used. weight/age (W/A), weight/stature (W/S) and stature/age Computer programs which allow image capture, the (S/A), using the NCHS tables.18 The z scores were calculation of the dimensions or number of objects divided into 2 nutritional status categories (well- designated by the user and the digital storage of this data nourished: z > 2DP, malnourished: z <-2DP), into three enable a precise and minute study of tissues, including the categories (well-nourished: z > -1DP, nutritional risk: z small intestine mucosa.6,7 Image capture enables analysis <-1DP to z -2DP and malnourished: z < -2DP) and were to be done by more than one observer, reduces the subjectivity also sorted in descending order. of the examination and facilitates the comparison of studies This was a retrospective cross-sectional study. The in this area. small intestine mucosa fragments were sliced and stained Morphometric studies of small intestine mucosa are with hematoxylin and eosin for histological study. Patients beginning to be used to evaluate the extent of alterations were included if their mucosa fragments, cut found in malnourished patients. Some of these studies perpendicular to the longitudinal axis of the tissue, had, reveal a clear correlation between nutritional status and at least six villi and six crypts for morphometric and small intestine mucosa alterations,8-11 while others have stereological evaluation, giving a total of 65 fragments. not corroborated these findings.3,12-15 However, the The new fragments were measured with the micrometer, morphometric techniques employed for this research are as in the earlier study. After images of the intestinal not uniform making comparison of the results difficult. In a mucosa had been captured, performed by means of a previous study,16 the morphometric analysis of the small microscope coupled to a computer, computerized intestine mucosa of 85 children, between 4 months and 10 morphometry was used to compare the two methods. The years old, was performed using a micrometer, coupled to an small intestine mucosa variables studied, by micrometer optical microscope which was the only method available at and digital morphometry, at 100 times magnification, the time. The data analysis, using the Spearman correlation were: the height of the villi, the depth of the crypts, coefficient, showed a positive correlation between: the mucosa thickness, total mucosa thickness and the villus/ height of the villi and the z score W/A; the total mucosa crypt ratio (Figure 1). Additionally, computerized thickness and W/A and W/S z scores and between the villus/ morphometry was performed of the average height of at crypt ratio and W/A and W/S z scores. least 30 enterocytes, of the nuclei of the enterocytes and In the present study, the use of a computer enabled of the brush border (Figure 2), at 500 times magnification. capture and storage of images of the small intestine mucosa, The cells selected had a basal nucleus and were located and also computerized morphometry of a proportion of the in the central third of the villus. The average height of material that had been used previously. Furthermore, each variable was calculated, dividing the sum of the capturing the images at 500x magnification allowed measurements by the number of units observed. measurements to be made of the height of the enterocyte, Additionally, in order to estimate the surface area of the the height of the nucleus and its brush border. In order to small intestine mucosa fragments, stereological analysis study possible alterations to the mucosa surface the was performed by means of a count of cycloid curves, Digital morphometric and stereologic analysis... – Pires ALG et alii Jornal de Pediatria - Vol. 79, Nº4, 2003 331

was an absolute number. The estimate of surface area was made using the formula:

Sv = 2. (I/ L) I = intersection: the sum of the number of times that the cycloid curve and the points touched the epithelial surface; L = total length of the test lines; i.e. it’s the entire grid and it’s a constant: 4.9297mm.

Morphometry was performed by the first author of the study, blind to the nutritional status of the patient. In order to capture images of the small intestine mucosa, a conventional optical microscope was used coupled to a video camera linked to a microcomputer with a video capture board and image analysis software. The images were captured as tag image format. The software used for digital morphometric analysis was Scion Image (Rasband 1. Top of the villus 1 – 2 Height of villus W. Scion Image Beta2 on line version at: http:// 2. Villus/crypt junction 2 – 3 Depth of the crypts www.scioncorp.com ed: National Institutes of Health, 3. Lamina propria 1 – 4 Total mucosa thickness 4. Muscularis mucosae 2 – 4 Mucosa thickness modified by Scion Corporation - Frederick, MD, USA; 1997).17 Figure 1 - Characteristics assessed in the small intestine mucosa For the statistical analysis, the quantitative data was through micrometer and computerized morphometry, initially described as mean average and standard deviation. at 100 times magnification Comparisons between two groups were made with the Student t test, for independent samples, with estimates of average difference and the 95% confidence interval (CI). In considering three groups analysis of variance (ANOVA) of from images captured at 40 times magnification (Figure classification criteria was used with significant differences 3). In order to measure surface area the number of times identified by the Student-Newman-Keuls post-hoc test. that the cycloid curve intercepted the epithelial surface Evaluation of linear correlation was made using the Pearson was counted and the subjacent tissue was evaluated by (r) correlation coefficient. The significance level adopted counting the number of points which fell between the was α = 0.05. Data was processed and analyzed using SPSS, epithelium and the lamina propria. The resulting number version 10.0 and Sigma Plot v. 2.1.19

Figure 2 - Photomicrography of the small intestine mucosa, stained with hematoxylin, at 500 times magnification. Variables studied: (1) height of the enterocyte, (2) height of the nucleus, (3) height of the brush border 332 Jornal de Pediatria - Vol. 79, Nº4, 2003 Digital morphometric and stereologic analysis... – Pires ALG et alii

Figure 3 - Photomicrography showing the cycloid curves superimposed upon the small intestine mucosa, in order to estimate the area of the mucosa surface, stained with hematoxylin, at 40 times magnification

This study was approved by the Commission for Ethics follows: z W/A = 29 WellN and 36 MalN; z W/S = 41 in Research of the Research and Post Graduate Unit at the WellN and 24 MalN; z S/A = 45 WellN and 20 MalN. Hospital de Clínicas de Porto Alegre. Nutritional assessment into three categories showed: z W/ A = 17 WellN, 12 nutritional risk (NRisk) and 36 MalN; z W/S = 22 WellN, 19 NRisk and 24 MalN; z S/A: 28 WellN, 17 NRisk and 20 MalN. Five patients presented nutritional Results edema. The average values for the variables studied at 100 The average age of patients in this study was 1 year and times magnification, including all patients from the earlier 5 months (+ 9 months), 50 of them (77%) were less than 24 study16 and those from the current study are given in Table months old and Caucasians predominated. The average 1. The average values for the variables studied at 500 times duration of diarrhea was 5.3 (+ 5.4) months. There was no magnification, including 65 patients, were: height of the relationship between the duration of diarrhea and nutritional enterocyte: 28.7 + 4 µm; height of the nucleus: 8.1 + 1 µm; status. Nutritional assessment into two categories was as height of the brush border: 1.9 µm + 0.3 µm.

Table 1 - Comparison of the characteristics of the small intestine mucosa through morphometry measured with micrometer

Variable First study* Current study (85 patients) (65 patients)

Total mucosa thickness 485.1 ± 111.8 µm 491.7 ± 80.8 µm Height of the villi 268.3 ± 87.5 µm 286.6 ± 62.9 µm Depth of the crypts 112.9 ± 33.8 µm 103.9 ± 16.8 µm Mucosa thickness 210.6 ± 73.2 µm 205.5 ± 49.1 µm Villus/crypt ratio 2.7 ± 1 2.8 ± 0.7

* Reference 18 Digital morphometric and stereologic analysis... – Pires ALG et alii Jornal de Pediatria - Vol. 79, Nº4, 2003 333

Employing the Student t test, the morphometric analysis the enterocyte, height of the nucleus and of the brush border, revealed that when the patients were divided into two assessed across 65 fragments, taking into account the nutritional status categories, according to z score for W/A, malnutrition classification by W/A, when the t test was used W/S and S/A, there were no statistically significant the characteristic which presented the greatest difference differences between the variables height of the villi, depth between the well-nourished and malnourished groups was of the crypts, mucosa thickness and total mucosa thickness the height of the enterocyte (well-nourished: 29.8 µm, and the villus/crypt ratio, performed on 62 fragments. malnourished: 27.9 µm). The same was observed of the When the patients were divided into three nutritional status classification according to W/S (well-nourished: 29.4 µm, categories, the digital morphometric analysis revealed malnourished: 27.6 µm). Despite this, none of the differences statistically significant differences for the villus/crypt ratio achieved statistical significance. between well-nourished and those at nutritional risk and Nevertheless, when the Pearson correlation coefficient between well-nourished and malnourished. However, there was used to evaluate correlations between the different was no difference between mildly malnourished and digital morphometry parameters and the z scores, a clear moderately and severely malnourished patients. The villus/ association was observed between the height of the crypt ratio was greatest among well-nourished patients. enterocyte and the W/A and W/S z scores; between the In our previous study,16 the analysis performed using height of the nucleus and the W/A z score and between the the Spearman correlation coefficient showed a positive height of the brush border and the W/A z score, although the correlation between the height of the villi and the W/A ratio, strength of this association was of low to, at most, moderate total mucosa thickness and the W/A and W/S scores and magnitude. Table 2 contains the correlation matrix, showing between the villus/crypt ratio and the W/A and W/S ratios, Pearson linear correlation coefficients and statistical with lower values being returned for the more severely significance for digital morphometry parameters against malnourished children. In terms of the variables height of nutritional status assessment.

Table 2 - Correlation matrix, showing Pearson linear correlation coefficients and statistical significance for digital morphometry parameters against nutritional status assessment

Variable Classification Weight/Age Weight/Height Height/Age (z score) (z score) (z score)

Height of the villi - µm 0.06 0.13 -0.01 at 100 times magnification, n = 62 p = 0.631 p = 0.309 p = 0.924 Depth of the crypts, µm -0.03 0.04 0.02 at 100 times magnification, n = 62 p = 0.803 p = 0.783 p = 0.858 Mucosa thickness, µm -0.15 0.02 -0.20 at 100 times magnification, n = 62 p = 0.231 p = 0.855 p = 0.125 Total mucosa thickness, µm 0.08 0.24 -0.06 at 100 times magnification, n = 62 p = 0.551 p = 0.060 p = 0.638 Villus/crypt ratio, µm 0.13 0.13 -0.22 at 100 times magnification, n = 62 p = 0.314 p = 0.307 p = 0.867 Surface/volume -0.02 0.19 -0.14 at 40 times magnification, n = 62 p = 0.871 p = 0.128 p = 0.281 Height of the enterocyte, µm 0.25* 0.29* 0.16 at 500 times magnification, n = 65 p = 0.038 p = 0.019 p = 0.179 Height of the nucleus, µm 0.24* 0.16 0.23 at 500 times magnification, n = 65 p = 0.054 p = 0.188 p = 0.063 Height of the brush border, µm 0.26* 0.27* 0.23 at 500 times magnification, n = 65 p = 0.032 p = 0.030 p = 0.062

* Statistically significant results. 334 Jornal de Pediatria - Vol. 79, Nº4, 2003 Digital morphometric and stereologic analysis... – Pires ALG et alii

Stereological analysis with cycloid curves, performed morphometry by micrometer, is probably a result of the across 62 fragments, did not reveal any statistically exclusion of 20 patients which included those older than 5 significant results in terms of nutritional status. and those whose fragments produced less than six villi and six crypts for morphometric analysis. In relation to the study of enterocytes, when the height Discussion of these cells was measured in 19 controls, children with no Persistent diarrhea occurs most often during the first diarrhea (32.3 + 3.8 µm) and 23 with persistent diarrhea two years of life.20 Of the patients in this study, 77% were (31.5 + 5.5 µm), without mentioning nutritional status, less than two years old, confirming the vulnerability of this Penna et al.25 report that no differences were detected age group to increased diarrhea prevalence. For Lentze,20 between the groups. However, a comparison between 24 it is difficult to distinguish which came first, the diarrhea or patients with normal histology and 18 with partial villi the malnutrition when dealing with malnourished children. atrophy (34.1 + 3.4 µm and 28.9 4.7 µm, respectively), statistically significant differences between the groups were While it is known that luminal nutrients are essential to found. Bhan et al.10 evaluated the enterocyte heights of the integrity of intestinal mucosa, the role played by different groups of children: well-nourished, with protracted malnutrition in the pathogenesis of malabsorption and diarrhea of varying etiologies, with coeliac disease and intestinal enteropathy remains controversial. Steatorrhea severe malnourishment. The values for enterocyte height and villi atrophy have been described in children with were greater among the well-nourished subjects (31 + 2.7 kwashiorkor and marasmus. The milder forms of malnutrition µm) and, in descending order, among those with severe do not appear to have a greater impact on the structure and malnutrition (25.3 + 1.7 µm); the enteropathogenic E. Coli function of intestinal mucosa and, probably, do not explain carriers (20.9 + 2.6 µm) and those with untreated coeliac the alterations to the mucosa architecture found in the disease (15.5 + 2.6 µm). Bahn et al.,10 in common with this tropics.21 The alterations described with patients suffering study, found higher values for enterocyte height among from protracted diarrhea, malnourishment or tropical well-nourished subjects when compared with severely enteropathy, are particularly related to alterations to the malnourished patients. morphology of the mucosa, the mucosa surface area and enterocytes. Kallas et al.,22 comparing children with diarrhea The values observed in this study are similar to those due to classic enteropathogenic Escherichia coli with those found by Penna et al.25 in patients with partial villi atrophy, suffering from environmental enteropathy, observed higher and also those detected in a study of adult patients by Wood values in the crypt compartment amongst the environmental et al.26 who found statistically significant differences enteropathy sufferers. In Venezuela, Romer et al.,23 studying between Caucasian English patients (30.2 µm) Indian the small intestine mucosa of 24 children with diarrhea and immigrants (28.3 + 2.7 µm) and Afro-Caribbeans (27.2 + varying degrees of malnutrition, grouped according to the 2.7 µm). The authors proposed that these findings “..reflect Gomez classification system, did not observe a correlation the ‘normal’ morphology of immigrant patients, determined between morphological alterations and the degree of by environmental factors “. malnutrition. Ferreira and Fagundes Neto,24 in São Paulo, When the enterocyte size of the patients in this study found alterations of varying intensity to the intestinal mucosa were analyzed with the Student t test in terms of W/A and of children with protracted diarrhea which were not related W/S z score classifications, there were greater differences to their nutritional status. Sullivan15, assessing the jejunal between well-nourished and malnourished patients, although mucosa from the small intestines of malnourished children these never achieved statistical significance. However, (W/S < 75% of NCHS), found a spectrum of alterations: analysis by means of the Spearman linear correlation from normal mucosa to mucosa similar to that observed coefficient revealed statistically significant differences with coeliac disease sufferers. It was further observed that, between enterocyte height, height of the nucleus and height 29 of them had marked crypt hypertrophy, with an elevated of the brush border and the W/A ratio and between enterocyte intraepithelial lymphocyte count. In our earlier study,16 we height and height of the brush border and the W/S ratio. found a positive association between W/A and W/S z There is a tendency for the height of these structures to scores, and, in decreasing order, measurements of villi reduce in proportion to the extent that the degree of height, total mucosa thickness and the villus/crypt ratio malnutrition increases. This finding suggests that which were lower in those that were most malnourished. malnutrition per se may be associated with the reduction in The crypt depth, however, did not reveal any differences enterocyte height. Reductions in enterocyte nucleus and between the groups. In the current study, which encompassed brush border measurements were also found among the 65 of those children, we added an assessment of nutritional most malnourished patients. status which included nutritional risk and found a positive Stereological analysis to measure the absorbent intestinal association of the villus/crypt ratio between well-nourished surface area, performed by means of cycloid curves in this and those at nutritional risk and between well-nourished study, did not reveal a positive correlation with nutritional and malnourished, with malnourished patients having lower status. Ribeiro et al.,27 however, in an experimental study of values. The differences between the two studies, in the epithelium of the small intestine using rats with protein- Digital morphometric and stereologic analysis... – Pires ALG et alii Jornal de Pediatria - Vol. 79, Nº4, 2003 335 energy malnutrition, found reduced jejunal absorbent surface 6. Hamilton PW, Allen DC. Morphometry in histopathology. J area while there was no observable alteration to villi height. Pathol 1995;175:369-79. They concluded that malnutrition can affect intestinal 7. Rahman SM, Itakura H. Morphometry in histopathology. An image analysis workstation for pathology laboratory. Anal Quant function by reducing adsorbent surface area with no apparent Cytol Histol 1996;18(6):471-80. 26 structural alterations. Wood, Gearty and Cooper, as in 8. Stanfield JP, Hutt MS, Tunnicliffe R. Intestinal biopsy in our research, also found no statistically significant difference kwashiorkor. Lancet 1965;2:519-23. in terms of surface area/volume, when studying Indian, 9. 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26. Wood GM, Gearty JC, Cooper BT. Small bowel morphology in 29. Fogaça HS. Desnutrição. In: Elia CCS, Souza HSP. Imunologia British Indian and Afro-Caribbean subjects: evidence of tropical da Mucosa Intestinal. São Paulo: Atheneu; 2000.p.159-63. enteropathy. Gut 1991;32:256-9. 27. da Costa Ribeiro H Jr., Teichberg S, McGarvey E, Lifshitz F. Quantitative alterations in the structural development of jejunal Corresponding author: absorptive epithelial cells and their subcellular organelles in Ana Luiza Guedes Pires protein-energy malnourished rats: a stereologic analysis. Rua Dr. Tauphick Saadi, 357/602 Gastroenterology 1987;93:1381-92. CEP 90470-040 – Porto Alegre, RS, Brazil 28. Madi K, Zaltman C, Takiya CM. Arquitetura da mucosa intestinal e sua plasticidade. In: Elia CCS, Souza HSP. Imunologia da Tel.: +55 (51) 3331.2657 / Fax: +55 (51) 3333.3000 Mucosa Intestinal. São Paulo: Atheneu; 2000.p.11-39. E-mail: [email protected]