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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2018 Sep 2;99(4):189–198. doi: 10.1111/iep.12286

A histomorphometric classification system for normal and inflamed mouse duodenum—Quali‐quantitative approach

Airton Pereira e Silva 1,2, João R A Soares 1,3, Erika Bertozzi de Aquino Mattos 1,2, Claudia Josetti 1,2, Isabelle M Guimarães 1,3, Sylvia M N Campos 1,4, Gerlinde A P B Teixeira 1,2,3,✉
PMCID: PMC6157301  PMID: 30175413

Summary

Gut‐associated intestinal lymphoid tissue, the largest secondary lymphoid organ in the human body, constantly samples antigens from the gut lumen, presenting as a default response the activation of TCD4+ FOXP3+ regulatory T cells that secrete a profile of anti‐inflammatory cytokines maintaining gut homeostasis denominated from an immunological perspective as mucosal tolerance. However, when antigens are sampled in an inflammatory setting, the immune response may either be protective, in the case of harmful pathogens, or cause further inflammatory reactions as in food allergy, inflammatory bowel diseases, coeliac disease or food protein‐induced enterocolitis syndrome. Therefore, there is a need for accurate and consistent experimental models. However, a drawback in comparing these models is the lack of a classification system similar to that which is already used for humans. Thus, the aim of this work was to propose a classification system of the small intestinal histomorphology in experimental mice. To do this we used a mouse antigen‐specific gut inflammation model developed by our research group. Duodenum sections stained with haematoxylin‐eosin and Alcian blue were scanned using the APERIO scanning system and analysed with the ImageScope® software. The evaluated parameters were villus area, villus height and width, enterocyte count, mononuclear intra‐epithelial leucocyte and goblet cell counts, and architectural and cellular ratios. Food‐sensitized animals challenged with a diet containing the corresponding food allergen presented, as for humans, time‐dependent shortened and widened villi accompanied by leucocyte infiltrate and loss of goblet cells. With these data, we were able to establish a classification system for experimental intestinal inflammation in mice thus permitting better comparisons among and between experiments than has been possible previously.

Keywords: food allergy, gut inflammation, inflammation score, mouse

1. INTRODUCTION

Gut‐associated intestinal lymphoid tissue (GALT), the largest secondary lymphoid organ in the human body, constantly samples antigens from the gut lumen.1 The default response is the activation of TCD4+CD25+FOXP3+ regulatory T cells (Tregs) that actively interact with antigens, derived from food or commensal bacteria, inducing the secretion of a profile of antiinflammatory cytokines that maintain gut homoeostasis. This tolerance to microbiota and food is also denominated mucosal tolerance.2 However, when antigens are sampled in an inflammatory setting, the immune response may either be protective, in the case of harmful pathogens,2 or cause further inflammatory reactions as in food allergy, inflammatory bowel diseases (IBD), coeliac disease (CD), food protein‐induced gastrointestinal diseases (FPIGD) or food protein‐induced enterocolitis syndrome (FPIES).3

Modern life, especially for children, has been accompanied by a rise in food allergy that is defined as an adverse reaction to food protein mediated by the immune system.4 Obviously, the main route of exposure to food allergens is the gastrointestinal tract; however, edible plant and animal antigens may also gain entry to the organism through the skin5 or the respiratory tract.3, 6 In allergic children or adults, allergy may manifest by reactions such as dysphagia, food refusal, early satiety, regurgitation, nausea, vomiting, diarrhoea or constipation, abdominal pain, presence of mucus and blood in stools after intaking the corresponding food.7

The initial manifestation of the signs and symptoms depends on the type of food allergy and ranges from minutes, in IgE‐mediated anaphylaxis, to hours or days in reactions mediated by cells and IgG.8, 9 The most efficient form of food allergy control, and therefore therapeutic approach, is the exclusion of the allergenic protein from the diet.10, 11 However, despite all care, accidental ingestion can occur, triggering allergic crises and increasing the risk of complications.12

A variety of inflammatory gut alterations are hallmarks of CD, FPIGD and FPIES intensity, and these have been standardized for humans.13, 14, 15 To this day, there is no standard for experimental gut inflammation models, and various research groups developed their own classification scores.16 Most models induce small intestine inflammation either through pathological agents17 (eg Heligmosomoides polygyrus) or using transgenic mice18 (eg Roquin‐deficient mice), whereas our group targets to replicate the intestinal manifestations observed in humans allergic to food.19 The use of human classification scores prompted us to establish an equivalent classification system for experimental models of food allergy that is accompanied by small intestine inflammation. Therefore, the aim of this work was to propose a classification system of intestinal histomorphology for experimental mice, covering quantitative and qualitative aspects, that allows the standardization of the states of normality and a grading of inflammation of the mucosal architecture of the mouse small intestine.

2. MATERIALS AND METHODS

2.1. Animals

Adult (12 weeks old, weighing ~25 g), male inbred C57BL/6 mice (originally obtained from the Jackson Laboratory in the 1970s) bred at the Animal Facility of the Federal Fluminense University (Niteroi, RJ, Brazil) were maintained with free access to food and acidified water (pH 2.5) in polypropylene cages with stainless steel covers (temperature of 22°C, ~60% humidity and 12‐hours light/12‐hours dark cycle).

2.2. Ethical approval

This study was approved by the local ethical committee and was performed in compliance with the national animal welfare committee. It follows ARRIVE guidelines.20

2.3. Experimental protocol

A mouse model of gut inflammation developed by Teixeira19, 21 was used. Briefly, animals were block‐randomized (as described by Suresh22) into three groups (allergic, tolerant and control nonallergic). To induce tolerance, commercial mouse chow was removed and replaced with a diet composed exclusively of peanuts in natura during seven days prior to systemic sensitization. During this period, allergic and control groups were maintained on commercial mouse chow. The allergic and tolerant groups were then submitted to a systemic sensitization protocol consisting of two subcutaneous inoculations of 100 μg of crude peanut protein extract (PPE) with a 21‐day interval. Control nonallergic animals were inoculated twice with physiologic saline (pH 7.2). Adjuvant (1 mg of AlOH3) was added only to the first inoculation of all groups. A week after the booster, each group was subdivided into two subgroups. The first continued to receive commercial mouse chow while the second was submitted to a challenge diet containing exclusively peanuts for 30 days. The number of animals per group varied from five to seven, and Figure 1 shows the steps of the experimental protocol.

Figure 1.

Figure 1

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Experimental protocol. Mice of the tolerant group were given free access to peanuts in natura for 7 days (starting at Day −7 through Day 0) prior to systemic sensitization with 100 μg of crude peanut protein extract with adjuvant in the tolerant and allergic groups. Twenty‐one days later, all groups received a booster with only the protein extract. Control nonallergic animals were inoculated twice with physiologic saline. The groups were then further divided (n = 5‐7 per group), and the allergic group received a challenge diet containing only peanuts in natura for 30 days before the end of the experimental protocol.

2.4. Necropsy and gut segment collection

The experiment was ended on the 30th day of the challenge diet period, with an overdose of anaesthetics (60 mg/kg of xylazine + 350 mg/kg of ketamine; Sespo Industries®, Paulinia, Sao Paulo, Brazil) as previously described.21 After examining the peritoneal cavity, a 5‐cm segment of the proximal small intestine starting at the gastro‐duodenum junction was collected for histopathology. These were fixed with 10% buffered formaldehyde and stained with haematoxylin‐eosin (HE).

The slides were scanned using the APERIO ScanScope CS System® with a 20× objective lens. To evaluate the histological parameters, imagescope ® software (v11.2.0.780; Leica Microsystems GmbH, Wetzlar, Germany) tools were used with a 7.2 digital zoom for cell counting and tissue analysis along with a touch pen and a track pad (Apple Inc.®, Cupertino, CA, USA). The duodenum in each slide was identified by its morphological aspects, such as the presence of tall and leaflike villi and high density of Brunner's glands as previously described.23 Tissue samples were longitudinally oriented to allow better assessment of each villus alteration. Along with general tissue evaluation including the crypts, the histomorphometric parameters evaluated were as follows:

  1. Determination of villus area: The area was established by outlining the boundary of each villus and is expressed in μm2.

  2. Measurement of villus height and width: Height was established by measuring the distance between the base and the apex of each villus. The width was measured in the median region of each villus, and spans the two epithelial monolayers plus the lamina propria.

  3. Villus height/width ratio (H/W): The H/W ratio of each villus was established by dividing the value of its height by the value of its width.

  4. Counts of intestinal epithelial cells (enterocytes—IEC), mononuclear intraepithelial leucocytes (IEL) and goblet cells (GC): The total number of IEC, IEL and GC was counted per villus. These counts were performed in both sides of each villus monolayer.

  5. IEC/IEL ratio and IEC/GC ratio: The IEC/IEL and IEC/GC ratios were established dividing the number of IEC by the number of IEL or GC, respectively, for each villus.

2.5. Establishment of bowel inflammation classification system

The classification structure used for the histopathological diagnosis of gut inflammation in humans, proposed by Marsh24 and later modified by Oberhuber,14, 25 was used as the starting point to establish the Mouse Bowel Inflammation Classification System. Both Marsh and Oberhuber assessed each villus height, width, IEC and IEL and IEC/IEL ratio. To these parameters, we added area, GC, H/W ratio and IEC/GC ratio per villus. Thus, a total of nine criteria were used to elaborate a classification system for experimental progressive intestinal injury: height, width and area; IEC, IEL and GC counts; and H/W, IEC/IEL and IEC/GC ratios in each villus.

2.6. Statistical analysis

All data are expressed as group mean ± SD (standard deviation). One‐way ANOVA with Bonferroni posttest was used to determine the minimum significant difference using graphpad prism 6.0 by GraphPad Software Inc.® (GraphPad Software, Inc., La Jolla, CA, USA). P‐values <0.05 were considered statistically significant.

3. RESULTS

3.1. General state of the gut

Overall, inflamed animal duodenum presented mild and moderate hypertrophy of the epithelium compared to normal animals. Oedema was also observed in inflamed gut. We did not observe major alterations in inflamed animal jejunum and ileum (data not shown).

3.2. Villus area and villus height/width ratio

Whether eating chow or peanuts, nonallergic groups (controls) presented normal morphology of the mucosa. The average villus area was 2.3 × 104 ± 102.53 μm2, villus height was 290.94 ± 40.10 μm, and the average H/W ratio was 4.57 ± 0.71. Tolerant animals irrespective of the challenge diet did not differ significantly from controls, although those eating peanuts presented a slightly smaller area (2.1 × 104 ± 100.91 μm2), villus height (270.73 ± 49.52 μm) and H/W ratio (3.99 ± 0.40). Allergic animals, submitted to the challenge diet, presented a significant decrease in the mean villus area (1.2 × 104 ± 123.00 μm2, P < 0.001) and mean villus height (199.58 ± 20.43 μm, P < 0.01) without significant changes in the width leading to a significant lowering of the H/W ratio (3.17 ± 0.44, P < 0.01).

As data of all animals submitted to control diet irrespective of prior treatment did not differ significantly, we present the graphical data of the control group submitted to control diet and tolerant and allergic groups submitted to peanut challenge diet (Figure 2).

Figure 2.

Figure 2

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A, Mean values of villus area expressed as μm2. The allergic group showed a significant decrease in area in each villus when compared to the control nonallergic (P < 0.001) and tolerant (P < 0.05) groups. No significant differences were observed between the control and tolerant groups. B, Mean villus H/W ratio. The tolerant and control groups showed similar ratios, while the allergic showed a significantly lower ratio than the control group (P < 0.01). Minimum n = 7 animals per group. * = P < 0.05, ** = P < 0.01, *** = P < 0.001

3.3. IEC and IEL ratio

The mean IEC/IEL ratio of the control group was 24.35 ± 1.40, of the tolerant group was 18.43 ± 1.13 and of the allergic animals was 6.85 ± 1.28. Thus, after the challenge diet period, a significantly lower IEC/IEL ratio was observed when comparing both the tolerant (P < 0.01) and allergic (P < 0.001) groups to controls (Figure 3B). The variations in the IEC/IEL ratio were caused by fluctuations in the IEL counts, whereas the IEC numbers did not alter significantly, as shown in Figure 3A. The allergic group showed a significantly higher IEL number per villus when compared to the control and tolerant groups (P < 0.05). Although the tolerant group presented an increase, it was not significantly different from controls.

Figure 3.

Figure 3

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A, Mean IEC and IEL counts. The allergic group showed a significantly higher IEL count than the control and tolerant (P < 0.05) groups. The control and tolerant groups did not differ significantly. IEC/IEL ratio (right) per group. B, The allergic group showed a significantly lower ratio than the control (P < 0.001) and tolerant (P < 0.01) groups. The control and tolerant groups did not differ significantly. Minimum n = 7 animals per group. * = P < 0.05, ** = P < 0.01, *** = P < 0.001

3.4. Mean number of GC per villus and ratio between IEC and GC

The mean of GC in control nonallergic animals was 13.28 ± 1.19 and in tolerant animals was 11.34 ± 1.23 while challenged allergic animals presented a significant decrease in GC per villus, 4.12 ± 1.57 (P < 0.001), leading to a IEC/GC ratio of 14.47 ± 1.40 in normal animals, 16.87 ± 1.76 in tolerant animals and 22.48 ± 1.90 in the challenged allergic animals (Figure 4).

Figure 4.

Figure 4

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A, Mean values of GC per villus in each group. The allergic group showed a significantly lower number of GC/villus (P < 0.001) than the control and tolerant groups, which presented similar number to each other. B, Mean IEC/GC ratio per villus in each group. The allergic group showed a significantly higher ratio (P < 0.01) compared to the control group; P < 0.05 when compared to the tolerant group. Minimum n = 7 animals per group. * = P < 0.05, ** = P < 0.01, *** = P < 0.001

3.5. Bowel inflammation classification system

The parameters from the histomorphometric evaluation were used to stratify the alterations in the mucosal architecture. The option was to classify into 5 levels: normal, infiltrative reaction, partial destruction lesion, subtotal destruction lesion and total destruction, detailed below (Table 1).

Table 1.

Summary of the parameters used to propose the inflammation score

Parameters evaluated Intestinal mucosa status
Normal Infiltrative Partial destruction Subtotal destruction Total destruction
Villi per 4000 μm 15‐25 15‐25 10‐15 <10 1‐3
Area (μm2) 2.0 × 104‐2.5 × 104 2.0 × 104‐2.5 × 104 1 × 104‐1.8 × 104 <1 × 104 Parameters were unable to be measured due to massive tissue hypotrophy
Villus height (μm) 200‐240 200‐240 150‐190 100‐150
Villus width (μm) 50‐70 50‐70 50‐70 50‐70
H/W ratio 3.5‐4.0 3.5‐4.0 3.0‐3.5 <3.0
IEC/villus 130‐150 130‐150 100‐130 90‐130
IEL/villus 0‐4 5‐10 10‐20 15‐25
IEC/IEL ratio 20:1 to 40:1 19:1 to 10:1, 9:1 to 5:1 <5:1
GC/villus 11.0‐14.0 7.0‐10.0 3.0‐6.0. <3.0
IEC/GC ratio 10‐15:1 16‐21:1 22‐29:1 ≥30:1
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  1. Normal intestinal mucosa: 15‐25 villi per 4000 μm, mean area of 2.5 × 104 μm2, H/W ratio of 3.5‐4, IEC/IEL ratio between 20:1 and 35:1 and mean number of GC/villus of 11.0‐14.0.

  2. Infiltrative reaction: histomorphometric analyses that showed the number, area and H/W ratio of each villus similar to normal intestine, with a decrease in the IEC/IEL ratio from 19:1 to 10:1, the mean number of GC/villus from 7.0 to 10.0 and an increase in the IEC/GC ratio to 21:1. Increased lamina propria width is also observed, because of infiltrating leucocytes.

  3. Partial destructive lesion: histomorphometric analyses that presented 10‐15 villi in the duodenum, with an area of 1 × 104 to 1.8 × 104 μm2, H/W ratio of 3.0‐3.5, IEC/IEL from 9:1 to 5:1, mean number of GC/villus from 3.0 to 6.0 and an increase in the IEC/GC ratio to 29:1. In this level, hypertrophy of both the lamina propria and the epithelium is visible due to the large inflammatory infiltrate (increase in IEL number), even though we did not score mitosis in the duodenum.

  4. Subtotal destructive lesion: histomorphometric analyses that presented <10 villi per 4000 μm, with an area smaller than 1 × 104 μm2, H/W ratio lower than 3.0, IEC/IEL lower than 5:1, mean number of GC/villus lower than 3.0 and IEC/GC ratio ≥30:1. At this stage, morphological aspects of each villus (tall and leaflike) were substantially altered because of vast lamina propria hypertrophy and subsequently higher villus width.

  5. Total destructive lesion: parameters could not be established due to massive mucosal and submucosal hypotrophy.

Representative images of the histological aspects presented are shown in Figure 5.

Figure 5.

Figure 5

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Representative images of the small intestine stained with HE showing the intestinal milieu after the challenge diet. A, The control group showed an intestinal mucosa staged as normal. B, The tolerant group showed an intestinal mucosa staged as Type 1 or infiltrative. C, The allergic group presented intestinal mucosa staged between Type 2 and Type 4 or from partial destructive lesions to (D) total destructive lesion. Images were captured using the ImageScope® software with 20× zoom. Minimum of n = 7 animals per group [Colour figure can be viewed at http://wileyonlinelibrary.com]

The comparative analysis between the results obtained and the proposed classification allowed stating that the allergic animals that consumed peanuts resulted in alterations in the mucosal architecture and cellularity compatible with partial to total destructive lesion. Tolerant animals presented an intact architecture and cellularity of the mucosal coating epithelium with an increase in the IEL leading to a reduction in the IEC/IEL ratio and lamina propria increase, compatible with the intestinal infiltrative reaction (Figure 6).

Figure 6.

Figure 6

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Higher magnification (scale bar, 50 μm) of tolerant animal villus stained with HE. Arrows indicate the location of IEL [Colour figure can be viewed at http://wileyonlinelibrary.com]

4. DISCUSSION

Irrespective of the presence of a positive serology, intestinal histopathological analysis is still considered a determinant requirement for FPIGD diagnosis.13, 26 The most serious lesions usually occur in the proximal small intestine and tend to diminish in severity as they approach the large intestine.27 In the classification system proposed by Marsh,24 the subclinical stages of FPIGD were classified as Phase 1 (infiltrative type) and Phase 2 (hyperplastic type), while the stage where clinical signs and symptoms are present, Phase 3 (destructive type), was frequently observed. Later, Oberhuber14, 25 proposed a subdivision of Phase 3 into 3a, 3b and 3c, characterized by increasing damage to the mucosa. Similarly, Villanaccia28 proposed a subdivision of 2 grades: Grade A (subclinical or not atrophic) and Grade B (clinical or atrophic). Similar to Oberhuber, Villanaccia proposed a subdivision of the clinical phase (Grade B1 and Grade B2) to portray the increasing damage to the mucosa,29, 30, 31, 32 Rito.28, 32, 33

Our group has been working on an experimental model that mimics the natural history of food tolerance and allergy induction.34 To better understand the factors involved in the induction of tolerance and allergy and the inflammatory consequences of the challenge diet, we have performed variations of the original protocol established by Teixeira.34 Animals are either fed a new protein or left on the mouse chow they have been weaned with, prior to a subcutaneous inoculation of the allergen. Therefore, our main findings are focused on the duodenum, and no major morphological alterations were observed in other parts of the small intestine besides a small increase in the IEL count in the jejunum and ileum (data not shown), similarly to various diseases observed in humans, such as milk and soya protein allergies.35

As has been described by many authors since the early 1900s,36 a percutaneous inoculation of protein antigens to animals that have not eaten the corresponding protein renders them allergic or sensitized, while the same inoculation in animals that have eaten the corresponding protein makes them tolerant. Immunological tolerance is characterized by a lack of an inflammatory immune response leading to the classical definition of oral tolerance: a specific immunological unresponsiveness.37 However, in the past decades an increasing number of papers have been published showing that tolerance is not simply an unresponsiveness or suppression of the immunological response but an active response triggered by Tregs.8 The intestinal mucosa houses the largest percentages of Tregs, above the average 10% of the total CD4+ T‐cell population of the other tissues or organs. In the small intestine lamina propria, they constitute an average of 20% and almost a third of the CD4+ T cells in the colonic lamina propria.38

The regulation of the mucosal immune responses by the intestinal Tregs occurs as a multiple cellular and molecular mechanism.39 In the presence of Tregs, naive bystander T cells are inhibited to differentiate to TH1 or TH2 profiles maintaining immune tolerance to dietary components and intestinal microbiota. However, if TH1 or TH2 lymphocytes are present and the antigen is presented in the gut lumen, pro‐inflammatory reactions will occur.1

In this work, along with the assessment of clinical aspects during the induction of food allergy and gut inflammation, gut histopathology was performed. Similar to previous findings of our group, the results obtained led to an array of histological alterations spanning from normal histology (control animals) to severe inflammation of the duodenum (allergic group).19, 40, 41 The pathophysiological changes induced by the inflammatory response led to alterations both in the cell count and in the villus architecture. No animal presented classical anaphylaxis symptoms, such as convulsions, wheezing, laboured respiration or pilar erecti.42, 43

The variation in the number of IEL inserted among the cells of the intestinal epithelial tissue is an important marker of the physiopathology of the small intestinal mucosa. The IEC/IEL ratio is widely used to differentiate the homoeostatic from the inflammatory state of the gut.44, 45 Considering that the number of IEC did not alter significantly within our experimental groups, the variations in both IEC/IEL and IEC/GC ratios were caused by the change in IEL and GC numbers.

It is worth mentioning that, in normality, villus height varies according to anatomical position and that IEC counts correlate positively with villus height; thus, shorter villi have lower IEC count, and vice versa. However, during inflammatory states, villus height may not correlate with IEC number, as villi can change their shape.46 For example, even though villi tend to become shorter, many times they become wider, explaining the maintenance of the IEC count. However, in more intense inflammatory scenarios, when villi flatten, cell counts tend to be significantly lower.

Animals of the tolerant group presented a decrease in the IEC/IEL ratio due to an increased leucocyte migration to the duodenum villi with no other alteration either histopathological or clinical. This phenomenon has been previously observed by us. Considering that eating induces tolerance and that animals of the tolerant group differ from the normal control only by the fact that they received a subcutaneous inoculation of the antigen, our hypothesis is that this inoculation is probably activating systemic Tregs that migrate to the gut upon a challenge diet.

Allergic animals presented a time‐dependent damage to the duodenum mucosa characterized by a progressive flattening of the villi, an increased IEL count leading to a lower IEC/IEL ratio and a lower turnover of GC leading to an increased IEC/GC ratio. Depletion of GC has an important impact on mucosal integrity due to the proportional decrease in mucin secretion, with consequent impairment of the protective function.47, 48 GC are induced by the differentiation factors Hath1 (and its mouse homolog Math1) and KLF4, and previous studies have already shown that these factors are poorly activated in ulcerative colitis, leading to GC depletion.49, 50 Other studies have shown that activated TCD4+ cells can modulate goblet cell differentiation during infections via IFN‐γ secretion, which increases IEC proliferation, thus limiting GC maturation.51, 52 Therefore, although inflammatory cytokines were not measured in this work, they may play an important role in causing the GC depletion observed here.

Based on the gastrointestinal changes observed, we developed an inflammation score for experimental mice. As the histopathological findings observed in our experiments are similar to those observed in human FPIGD, we based our score system on those previously established by Marsh,24 Oberhuber14 and Villanaccia.28

Thus, a 5‐category classification system was developed for mice: the first one describes the normal intestinal mucosa observed in the control or normal animals; the second category (infiltrative reaction) depicts the intestinal mucosa of the tolerant animals that received a subcutaneous inoculation with the corresponding antigen or the initial phase of gut inflammation—normal mucosal architecture accompanied by slightly higher villi throughout the duodenum and an increase in IEL in the epithelia, thus showing hypertrophy of epithelium; the third and fourth categories describe the progressive alteration of the mucosal architecture with an increasing number of the IEL within the IEC layer, therefore presenting increasing hypertrophy of the epithelial layer; and finally, the fifth category shows a massive destruction of the duodenum. In our system, this only occurs when the animals are submitted to longer challenge diet periods (over 80 days). A strong correlation (data not shown) was observed between a major body weight loss and the massive hypotrophy of the mucosal and submucosal architecture.

It is important to mention, however, that once the allergen is removed from the diet, a progressive improvement can be observed in the small intestine. In our model, it takes at least three weeks for the intestinal mucosa to recover its normal architecture, although high IEL levels (when compared to control animals) still persist, as shown in previous works of our group.41

There are several experimental mouse models to study FPIGD. In a comprehensive review, Liu53 listed 26 models and their differences, such as sensitization and challenge routes. Similarly, Erben et al16 evaluated different mouse models of intestinal inflammation, including small intestine and colonic inflammation using chemically induced or genetically altered mice, while other authors evaluated small intestine histology alterations during challenge diet periods.54, 55, 56, 57 Although all of them observed inflammatory markers in the intestine, none of them performed a detailed and quantified analysis of the duodenum villi as presented in our work.

The duodenum inflammation score proposed here allowed a better diagnostic classification for the assessment of the dynamics and evolution of histological alterations caused by the experimental model used in our group.26 We can argue that this classification system can mitigate arbitrary scoring and permit the setting of standards among research groups. Further detailing and therefore a better classification can be obtained using immunohistochemistry to identify the dynamic and evolution of the inflammatory cells in the gut. In conclusion, a detailed experimental duodenum inflammation classification system was successfully established.

ACKNOWLEDGEMENTS

The authors would like to thank Professor Maira Platais for the English review.

Pereira e Silva A, Soares JRA, Mattos EBA, et al. A histomorphometric classification system for normal and inflamed mouse duodenum—Quali‐quantitative approach. Int J Exp Path. 2018;99:189–198. 10.1111/iep.12286

Funding information

This work was supported by the grant provided by FAPERJ Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro—“Carlos Chagas Filho Foundation for Supporting of Research of the State of Rio de Janeiro” under Grant Number E‐26/110/409/2011 and doctorate fellowship provided by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico—“National Council for Scientific and Technological Development”).

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