Ultrastructural changes in chronic inflammatory enteropathies—a comparison between dogs and humans

Chronic inflammatory enteropathies (CIEs) are an important group of diseases in dogs and involve complex pathogenetic aspects. Endoscopy and histopathology are vital for documenting the disease but are less useful for subclassifying CIEs and predicting the response to treatment. However, healing of the mucosal disease process (deep remission) and ultrastructural evaluation of the mucosa have received little attention in canine CIE. Given that canine CIE shares many similarities with inflammatory bowel diseases (IBDs) in human patients—and presents a good spontaneous disease model for human IBD—this perspective article evaluates the literature on ultrastructural lesions in canine CIE and human IBD and offers future directions for the study of ultrastructural mucosal lesions in canine CIE. Such lesions might have a higher sensitivity of detection than structural changes revealed upon light microscopy and may even precede or remain after the resolution of the clinical signs and histologic lesions.

a monitoring tool, especially as the histologic resolution of duodenal lesions in CIEs was absent in most dogs responding during the induction phase of treatment (Garcia-Sancho et al., 2007;Schreiner et al., 2008).However, the role of mucosal healing (deep remission) has not been extensively investigated in canine CIEs (Jergens and Heilmann, 2022).
Canine CIE shares many similarities with chronic inflammatory enteropathies or inflammatory bowel diseases (IBDs) in human patients (Jergens and Simpson, 2012), comprising Crohn's disease (CD), ulcerative colitis (UC), and indeterminate inflammatory bowel disease (Beniwal and Harrell, 2010;Ashton and Beattie, 2023).Despite these overlapping disease characteristics, several diagnostic and management features appear to be unique to either canine CIE or human IBD, including the disease location and distribution, immunological signatures such as the cytokine profiles of T helper cells (Heilmann and Allenspach, 2017), and long-term risks (Jergens and Simpson, 2012).However, canine CIE-despite presenting a good spontaneous disease model for human IBD-is generally less studied than human IBD.

Intestinal ultrastructure
The intestinal tract has the basic structure of a membranous-muscular tube.The luminal mucosa comprises an epithelium overlaying a basement membrane and a loose connective tissue layer (lamina propria mucosae).The underlying lamina muscularis mucosae consist of smooth muscle cells that facilitate the motility of the intestinal mucosa.In the small intestine, mucosal enlargement results from mucosal folds with characteristic fingerlike villi of approximately 0.5-1.6 mm in length that protrude into the intestinal lumen and tubular crypts that extend into the intestinal wall (Liebich, 2010;Kummer and Welsch, 2018).
Villi are generally absent on the mucosa throughout the large intestine, but the large intestinal mucosa contains small folds and deep tubular crypts at a high density.The villi and crypts are covered by a single layer of columnar epithelium, mainly consisting of absorptive enterocytes, goblet cells, and few endocrine (Paneth) cells.Enterocytes are characterized by an apical brush border consisting of microvilli.These microvilli have an approximate width of 80 nm, height of 1.0-1.4µm, and are covered by a 0.3-0.5-µmglycocalyx layer (Marin et al., 1983).However, the abundance of microvilli in the epithelial cells of the large intestine is significantly lower than that in the small intestine (Washabau, 2013).Enterocytes have an oval-shaped nucleus that is located in the basal ⅔ compartment of the cell and is overlaid by the Golgi apparatus, lysosomes, mitochondria, and rough and smooth endoplasmic reticulum (ER).Goblet cells ensure a continuous merocrine release of a glycoprotein-and glycolipid-rich cytoprotective mucin layer.These cells have a narrow base with a basal nucleus and organelles composed of a well-developed ER and Golgi apparatus.The apical part of the goblet cells is typically dilated and filled with mucin droplets, which are membrane-bound and 1-2 µm in diameter.
Epithelial cells are attached to the basal membrane via hemidesmosomes and are firmly linked together by an apical junctional complex composed of-from the apical to basal region-tight junctions (TJs) (zonula occludens), adherens junctions (AJs) (zonula adherens), and desmosomes (macula adherens), providing adhesive and other mechanical properties that seal and restrict the exchange of substances across the paracellular space (Toner, 1968;Demling et al., 1969;Roda et al., 2010;Henderson et al., 2011;Buckley and Turner, 2018).
The submucosa separates the lamina muscularis mucosae from the muscularis propria and contains loose connective tissue with blood vessels, lymphatics, adipose tissue, and scattered immune cells, as well as the submucosal plexus (Meissner plexus), a nerve plexus that controls the secretion and motility of the inner intestinal wall layers.The muscularis propria consists of the inner circular and outer longitudinal layers of smooth muscle cells and a few interstitial cells of Cajal that have an intestinal pacemaker function.Located between these two muscle layers is the myenteric plexus (Auerbach plexus) that regulates the peristalsis of the muscularis layer.The muscularis propria in most areas of the intestinal tract is covered by a single layer of flat epithelium, the serosa, which covers an underlying thin layer of connective tissue (Hoyle and Burnstock, 1989;Ibba-Manneschi et al., 1995;Liebich, 2010;Kummer and Welsch, 2018).

Ultrastructural disease characteristics of canine CIEs
Ultrastructural lesions can be expected to have a higher sensitivity of detection than structural changes revealed upon light microscopy and may even precede or remain after the resolution of the latter (Sbarbati et al., 2003).However, data on ultrastructural lesions in canine CIE are limited.Abnormalities of the brush border and mitochondrial lesions (cristeolysis and swelling) in endoscopic biopsies of the duodenum have been described in the food-responsive phenotype of canine CIE (Table 1) (Walker et al., 2013).Furthermore, these lesions improved upon clinical remission, showing a reduction in the enterocyte intermicrovillar space and increased microvillus height after 6 weeks of dietary intervention using a hydrolyzed protein diet (Walker et al., 2013).However, potential lesions in the ileum and/or

Qualitative observations a
Quantitative observations a

Ultrastructural lesions in human IBDs
Differential mucosal abnormalities in IBD (Table 2) included a preserved mucosal integrity with the loss of regular polygonal units and increased mucous production in patients with CD but sloughing and mucosal disintegration with decreased mucous production in UC patients (Trabucchi et al., 1986).Interestingly, these UC lesions could even be observed in endoscopically normal areas of the colon (Trabucchi et al., 1986;Bertini et al., 1998), making electron microscopy (EM) a sensitive tool to detect mucosal lesions and potentially deep remission.In other studies of human IBD, which focused on either CD or UC, the ultrastructural lesions evaluated included several alterations affecting the different structural units of the intestinal wall (Table 2) (Aluwihare, 1971;Ranlov et al., 1972;Dvorak et al., 1979;Myllarniemi and Nickels, 1980;Rickert and Carter, 1980;Lewis et al., 1984;Nyhlin and Stenling, 1984;Dvorak and Silen, 1985;Shields et al., 1985;Nagel et al., 1995;Bertini et al., 1998;Fratila and Craciun, 2010;Zhao et al., 2021;Zhou et al., 2023).While most lesions overlapped between childhood and adult cases of IBDs, some lesions presumed to reflect the chronicity of the disease process (e.g., villus bridging and goblet cell reduction) were primarily observed in adult IBD (Bertini et al., 1998) or were suspected to be specific for childhood IBD (Lewis et al., 1984).As an important differential diagnosis of IBD in humans, irritable bowel disease can also produce ultrastructural lesions (Miglietta et al., 2021).

Perspective
Lesion evaluation in human IBD and experimental animal models has traditionally focused on the enterocyte and typically includes the evaluation of the lesions that reflect changes in mitochondrial size and integrity, cytoplasmic injury, microvillus mass, and disruption of tight junctions (Mughal and Filipe, 1992).However, other structures of the mucosa might be equally important to be examined.Transmission EM (TEM) or scanning EM (SEM) could present a useful adjunct tool to evaluate the structures and detect lesions or patterns of tissue regeneration that remain undetected during routine histology (Laschi et al., 1987;Schattenfroh et al., 1994;Bertini et al., 1998).EM is an important tool in medical research and diagnostics, and this technique is usually accessible in most universities and research laboratories.
Very little information about ultrastructural lesions is available for canine CIEs (Walker et al., 2013).Morphological characteristics yet to be investigated in canine CIEs using TEM and/or SEM include any features and particularly goblet cells (and their subcellular mucin droplets) in the colon, any structural lesions in the ileum, mucosal structures other than the enterocyte, and a number of other enterocytic subcellular structures (e.g., ER, Golgi complexes, desmosomes, nuclei, and nucleoli) and cellular lesions including 1) junctional complexes (JCs) focused on TJs, AJs, and desmosomes; 2) autophagic bodies; 3) evidence of apoptosis or necrosis; 4) lipid/ chylomicron droplets; 5) other particles (including viral or phage structures); and 6) other lesions shown in human IBD (Table 2).In addition, ultrastructural changes in the response to treatment (or lack thereof) other than dietary intervention-including immunomodulatory treatment or alternative therapeutic options such as pre-/pro-/synbiotics, fecal microbiota transplantation, cholestyramine as bile acid sequestrant, or stem cell therapy (Jergens and Heilmann, 2022)-remain to be studied.In this regard, synbiotic treatment decreases the dispersion and size variation of microvilli and the disruption of enterocytes in a dog with CIE (Figure 1) (Sahoo et al., 2022).Given the number and complexity of possible ultrastructural alterations (Table 2), their detection, grading, and association with other patient and disease characteristics might benefit from using a machine learning Transmission electron micrographs of enterocytes lining the colon from a dog with chronic inflammatory enteropathy before (A) and after (B) treatment with synbiotics.Before synbiotic treatment (A), microvilli (MV) are dispersed and vary in size.Large vacuoles (V) show disruptions in the cytoplasm and distension of the rough endoplasmic reticulum (rer).Following synbiotic treatment (B), MV are densely uniform in size, and the enterocytes are markedly less disrupted.The nuclei (N), mitochondria (m), rer, and small vacuoles (v) appear normal.
algorithm.Deep machine learning might also allow researchers to identify and compare structural lesions along the gastrointestinal tract, follow and integrate longitudinal changes over a more extended time, and utilize an unsupervised convoluted neural network approach to identify currently underestimated or even undetected lesions (Syed and Stidham, 2020;Javaid et al., 2022;Zand et al., 2022).Despite the tedious preparative steps involved in electron microscopy (Laschi et al., 1987), this method shows potential to investigate and integrate still unknown or undetected aspects of canine CIE, especially in the context of its pathogenesis, diagnosis, and response to treatment.A better characterization and deeper understanding of the ultrastructural mucosal changes in canine CIE will ultimately lead to a better definition of the similarities and differences between human IBD and canine CIE and, thus, will shed more light on the suitability of the dog as a spontaneous animal model for human IBD.

TABLE 2 (
Continued) Summary of the ultrastructural lesions identified in human CD and UC.

TABLE 2 (
Continued) Summary of the ultrastructural lesions identified in human CD and UC.