As for age distribution among patients, ultrasonographic abnormalities were detected most frequently in their middle or later age. This corresponds to previous reports [23
], though the exact reason why middle-aged and senior dogs tend to suffer from gallbladder disorders is yet to be investigated. Toy or miniature breeds were overrepresented in our study. Whether this is a simple reflection of demography of dog breeds in Japan or is related to other factors needs further investigation. Neutered dogs accounted for almost 90% of the entire study population. These results differ from a previous report, in which no significant skewness was identified between the presence of sludge and the sex of the dogs [23
]. On the other hand, another report that described canine cholangitis, cholangiohepatitis, and gallbladder diseases showed a similar sex composition to ours in their 54-dog cohort [19
]. A similar tendency was noted in other 45-dog and 27-dog cohorts, respectively [6
]. The overt predominance of females as well as neutered individuals in several cohorts might indicate hormonal and/or metabolic effects on gallbladder disorders and indicates the need for additional case-control studies to know the basis for this result.
Ultrasonographic abnormalities of the gallbladder have been vigorously investigated [6
]. Clinical planning of veterinary clinicians for gallbladder mucocele seems to be well standardized and is dependent on specific ultrasonographic features detailing the disease stage and concurrent clinical information [30
]. Though sludge in the gallbladders of dogs has been regarded as an incidental finding without need for treatment [4
], 9 out of 24 dogs with mobile sludge or precipitate experienced gallbladder rupture in a previous study [6
]. Furthermore, a recent report by Saunders et al. discusses the merit of early cholecystectomy in dogs with gallbladder sludge, emphasizing a possible relationship of sludge and mucocele and minimizing a chance of complication during cholecystectomy [26
]. It is premature to make a conclusive statement on the best clinical approach for canine gallbladder sludge for the time being. In addition, as DeMonaco and colleagues [20
] and Secchi’s group [23
] have reported, a mere one-year follow-up of a spontaneous course of biliary sludge may not be long enough to fully understand its true nature as disease.
In prior research on animal gallbladders, detection of intralesional bacteria was attempted on various specimens using a variety of methods such as cholecystocentesis, direct sampling of the liver or gallbladder tissue during laparotomy/laparoscopy, or histochemistry (Gram stain) on archived histologic specimens [12
]. Fluorescence in situ hybridization has also been demonstrated to be a sensitive method to detect bacteria in mucocele specimens [33
]. In our study, culture of the gallbladder tissue or intracystic bile was seldom performed by the attending veterinarians, likely because of financial issues. We chose Giemsa and Warthin–Starry stains because they are cost-effective for the detection of bacteria in histology sections. The hurdle we encountered, however, was frequent leakage and loss of especially liquid gallbladder contents during tissue trimming. In addition, we were unable to speciate the pathogens by histomorphological observation alone. The past reports on bacterial isolation from dogs with hepatobiliary diseases included intestinal bacteria such as Escherichia coli
sp., and Clostridium
]. Whether these bacteria are involved with initiation and progression of cholecystitis, however, has long been controversial. Since we have not been able to track periodic changes of the gallbladder except by imaging studies, reproducible cholecystitis models and new methods to trace histological/cytological changes in gallbladders are needed. Future investigation using primary cell culture of the canine gallbladder mucosal epithelial cells, such as that developed by Oda and colleagues [34
], would increase our understanding on the response of gallbladder tissue against infectious agents.
Gallbladder contents can be roughly divided into two categories: mucocele and non-mucocele. While mucocele has been a target of a series of vigorous investigations, [11
] non-mucocele contents have gained little attention in veterinary medicine. Sludge in our cohort was histologically composed of fragmented gelatinous mucus, microliths, cell debris, and bacteria. Mucus that we microscopically detected in a majority of specimens resembled mucocele in their staining features (amphophilic to pale basophilic) and texture (homogenous and amorphous). This was consistent with previous work by Mizutani and colleagues, who clarified by infrared spectroscopy that both mucocele and sludge were equivalent to swine mucin in their chemical properties [12
]. Microliths, on the other hand, are aggregates of a brown, soft, granular substance interpreted as condensed bile in our study. Microliths grossly look very much like brown-pigmented human gallstones though they are typically formed in the bile ducts, not in the gallbladder as seen in our canine cohort [37
]. Human brown-pigmented gallstone is associated with biliary infection, which is likely the case in our study [37
]. Further investigation is necessary to determine the nature of microliths and if these are actually premature gallstones.
Mucocele, in our study, frequently occurred along with mild cholecystitis. This result is consistent with the findings in a previous work by Aguirre and colleagues [1
]. As Mizutani’s group pointed out, a sequential development from sludge-forming gallbladder disease to mucocele is a possible scenario [12
]. Tsukagoshi and colleagues also reported that postprandial gallbladder emptying was significantly reduced in those dogs with mobile/immobile sludge or mucocele when compared to control dogs without sludge or mucocele, suggesting that biliary stasis is an important pathologic basis for gallbladder diseases in general [24
]. In order to confirm that there is a transition from sludge-forming cholecystitis to mucocele, sequential evaluation on changes occurring in the gallbladder tissue in vivo, which does not seem feasible now, would be necessary by novel in vitro techniques.
A comprehensive and detailed description on cholecystitis in canine gallbladders has not been reported, except for the work by Lawrence and colleagues describing as few as six gallbladders [39
], as well as the report of Viljoen’s group on just 14 gallbladders [25
]. The present report is the first among the similar kinds that board-certified veterinary pathologists have conducted the comprehensive histomorphological evaluation of diseased canine gallbladders. In humans, on the other hand, histopathology of cholecystitis has been well documented [37
]. For example, in humans, cholecystitis is divided into acute and chronic forms [37
]. Each form includes calculous (gallstone-laden) and acalculous (no gallstone) cholecystitis [37
]. Human chronic “acalculous” cholecystitis has subclasses such as lymphoeosinophilic, eosinophilic, granulomatous, diffuse lymphoplasmacytic, and lymphocytic cholecystitis [37
]. Some chronic human “calculous” cholecystitis cases have minor components of plasma cells, eosinophils, macrophages, and neutrophils [37
]. “Calculous” versus “acalculous” nomenclature in veterinary medicine needs clarification to determine whether microliths are precursors of gallstones. Canine microliths grossly and microscopically resemble brown-pigmented gallstones of humans, but we need further physical and chemical research to clear this point. Fixing a nomenclature and disease classification is important to facilitate crosstalk between medical and veterinary medical experts as well as to support clinical decisions of veterinary practitioners.
Lymphoid follicles have not previously been reported in canine gallbladders. Prolonged antigenic stimulation seems to predispose this, but the precise etiology is yet to be investigated. In humans, follicular cholecystitis (FC) has been diagnosed by detecting three or more distinct lymphoid follicles per centimeter, and is associated with older age [40
]. Saka et al. reported that FC did not seem to be associated with autoimmunity, lymphoma, or obstructive gallbladder diseases in an investigation of 2550 human cholecystectomy specimens, in which there were only five samples fulfilling the diagnostic criteria of FC [40
]. We may need to refrain from using the term FC for canine gallbladders until there is a consensus on terminology among experts.
Contrary to the frequent isolation/detection of bacterial pathogens, neutrophilic inflammation was not frequently identified in canine gallbladders in our study and in the studies of others [25
]. This finding corresponded with the previous report on porcine chronic cholecystitis, in which lymphoplasmacytic inflammation was associated with the frequent detection of bacterial bacilli by use of the Warthin–Starry stain [41
]. Contrary to this, neutrophilic inflammation was much more prevalent in the livers of those dogs suffering from gallbladder diseases [19
]. Gallbladders with prominent neutrophilic infiltration in our canine population, though their number was low, seemed to represent acute-on-chronic inflammation, in which peracute secondary bacterial inflammation overlaid a pre-existing chronic lymphoplasmacytic cholecystitis.
IHC revealed the composition of lymphoid cells in 12 G2 lesions of our cohort. B lymphocytes predominated in the lamina propria, and a few plasma cells and fewer T lymphocytes were also seen. Granzyme-B-positive cells, suggestive of cytotoxic T lymphocyte or natural killer cell origin, were not detected in our study. Taken together with the detection of lymphoid follicles in 68 tissues (48 of G2; 20 of G1), there likely was a long-standing stimulation to the mucosal tissue by as yet unknown antigenic elements such as bacterial or bile-related constituents. The frequent detection of plasma cells around follicles was intriguing. This suggests increased production of immunoglobulin in this tissue. Since gallbladder mucin and immunoglobulin G have shown to accelerate nucleation in the development of gallstones, B-cell-dominant inflammation with plasma cells might be a research target to understand the mechanism of sludge/gallstone formation [38
]. Future studies from the standpoint of immunology are definitely needed.
A detailed histological evaluation of the thickened gallbladder wall has not been performed though it is a common abnormality observed in ultrasonographic examination of diseased gallbladders [6
]. Crews and colleagues documented intramural necrosis, hemorrhage, vascular thrombosis, inflammation, fibrosis, and mucosal hyperplasia in their 45-dog cohort; however, all of their subjects had gallbladder rupture, unlike our cohort [6
]. Our investigation was the first to describe the entire gallbladder wall with great detail, including measurement of mucosal thickness (unpublished data) and GWTT. The average (1071.9 µm) and median (859.5 µm) of GWTT obtained by our study was larger than that of normal dogs, in which GWTT rarely exceeds 500 µm (observation in routine histopathology by the authors). Contrary to previous reports, thickening of the gallbladder wall in our chronic cholecystitis cases was caused by mucosal hyperplasia, smooth muscle thickening, fibrosis, edema, congestion, lymphatic dilation, and hemorrhage. Smooth muscle thickening was associated with myocytic hyperplasia/hypertrophy, suggesting increased mechanical burden during gallbladder emptying, abnormal nerval inputs, or an effect of growth factor(s). Gallbladder wall edema has been reported in canine patients with pulmonary hypertension or anaphylaxis, implying an effect of increased static pressure or increased vascular wall permeability [42
]. The underlying pathogenesis for cholecyst wall edema, in our cases, likely differs in each case and may represent concurrent inflammatory status and/or vascular wall physiopathology. Intramural fibrosis of the gallbladders in our cohort was likely related to tissue repair during the chronic inflammatory process.
Intracytoplasmic accumulation of amber to light brown, fine granular pigments has not previously been reported in canine gallbladders. Gilloteaux and others conducted ultrastructural studies on chronic human cholecystitis and found osmiophilic lipofuscin-like bodies and lipomucosomes (a fusion of lipid deposits and mucus-containing vesicles in cholecystocytes) [44
]. These intracellular, as well as extracellular, structures led these researchers to hypothesize that intracellular components are released outside the cell by the detergent-like action of bile on cholecystocytic membranes, which then become aggregated to form biliary sludge [45
Mineralization of mucus, typically within mucosal crypts, as in our cases, has not been described previously in canine gallbladders to the authors’ knowledge. There was a microscopically detectable transition between bland intracryptic mucus and mineralized crystals; therefore, a calcium-binding property of mucus is speculated. The detailed mechanism behind it, however, should be clarified through investigations such as that of Imano et al., who identified osteopontin, a calcium-binding protein, in gallbladder epithelial cells and intralesional macrophages by IHC [46
Seventy-six dogs in our cohort had hepatic inflammation of a varied nature and degree, which is consistent with previous reports [18
]. When a patient presents with bacterial hepatitis, it must be determined whether it is caused by ascending or hematogenous bacterial entry. When it comes to cholecystitis, we also have to determine whether the introduction of bacteria occurs by ascending or hematogenous entry. For cholecystitis of animals, the frequent detection of alimentary bacterial species supports the ascending infection theory [6
]. Differences in pathologic features of gallbladder diseases between cats and dogs should also be taken into consideration [28
]. In particular, bacteremia, not bactibilia, should be closely examined in future studies to clarify the entry route and exact roles of alimentary pathogens involving hepatobiliary diseases.
Hepatic lobular diameter (HLD) is an objective indicator to assess microhepatica, pathognomonic findings for a hepatic vascular anomaly such as PPVH and portosystemic shunts in dogs [31
]. The reason we measured HLD in our study was because we hypothesized that a decreased mass of liver (decreased number of total hepatocyte) may lead to a decreased net volume of a hepatocyte-derived, yet-unknown protective element for a gallbladder mucosal epithelial cell. If we can correlate PPVH (diagnosed by a combination of “decreased” HLD and other already-mentioned histologic findings [31
]) with gallbladder abnormality, the liver, as a producer of various physiologic elements, can be an important and novel target for further research. Our results, however, did not show a statistically significant relationship among decreased HLD, mucocele, and sludge. This result indicates that PPVH, a frequently diagnosed nonlethal condition in miniature or small breed dogs, is less likely a contributor for the development of gallbladder disorders.
Hepatocellular degeneration was detected in almost all dogs in this study, supporting the frequent previous detection of increased serum liver enzyme activities. Severity of degeneration, however, was typically mild among our subjects. Those dogs with more severe hepatocellular degeneration often had concurrent significant inflammation in portal tracts. In previous reports, hepatocellular degeneration of varying degree and nature was also detected in canine patients with gallbladder diseases, but a precise description of each liver component (portal tract, parenchyma, capsule, and so on) was not available [1
The cause of chronic cholecystitis is an enigma. There have been a handful of opinions with/without supportive evidence regarding this disease. Frequent isolation/detection of alimentary bacterial species from gallbladder contents/tissues has led to a deeply entrenched notion that cholecystitis is most likely derived from bacterial infection. It may be true, but we have not been able to reproduce cholecystitis by artificial bacterial infection alone. Research by Kaminski et al., using feline subjects, suggested the possible role of arachidonic acid metabolites on the development of cholecystitis, but it has yet to be proven that this applies to cholecystitis in other animal species [48
]. Kakimoto and colleagues have attributed canine gallbladder diseases to altered bile acid composition [22
]. Ultrastructural work by Gilloteaux et al. suggested a possible involvement of altered lipid metabolism, which causes a subcellular accumulation of lipid deposits and a resultant sloughing of cholecystocytes (gallbladder mucosal epithelial cells), leading to the formation of bile sludge [45
]. Hence, at this point, it would be safe to say that chronic cholecystitis seems to be a disease of multifactorial etiology. Similarly, there has been much speculation on the factors contributing to gallbladder mucocele, which is beyond the scope of our study [8
]. Kesimer and colleagues performed an extensive analysis of canine gallbladder mucus, including the physical, chemical, and functional features of mucus, in their study of mucus hypersecretion in canine gallbladders [35
]. This would be a powerful approach to the study of chronic cholecystitis pathobiology as well.
Lastly, our statistical analysis was designed to determine whether null (G0), mild (G1), or severe (G2) gallbladder inflammation was related to any ultrasonographic or clinicopathological parameters. Those parameters showing statistical significance between G0 vs. G1 or G0 vs. G2 were judged to be useful in differentiating between an “inflamed gallbladder” and an “uninflamed gallbladder”. Such parameters included mucocele, GWTT, bacteria, lymphoid follicles, smooth muscle thickening, and edema. Among these features, smooth muscle thickening and edema could be a more sensitive indicator of gallbladder inflammation than the rest because these two parameters showed statistical significance even in the pair of G0 vs. G1, while others did so only in G0 vs. G2 pairing. On the other hand, those parameters showing statistical significance between G1 vs. G2 were judged to be of some use in predicting the “severity” of cholecystitis. Parameters associated with severity were sludge, GWTT, bacteria, lymphoid follicle, smooth muscle thickening, and liver inflammation. We also examined the relationship between sludge or mucocele and various clinicopathological parameters. The results showed that mucocele was mutually exclusive with sludge, edema, and PPVH. On the other hand, mucocele was related to increasing values of age, GWTT, and HLD. Most of these results are intuitive and supported by histological features. PPVH does not seem to predispose the patient to mucocele formation, so mucocele is likely a disease independent of liver abnormality. As for sludge, our analysis revealed a mutual exclusion of sludge with GWTT or liver inflammation. Therefore, gallbladders with normal wall thickness could have undergone a pathologic process. In addition, sludge can be caused by a process indifferent to the inflammatory condition of the liver. All these findings based on statistics, however, should be interpreted and extrapolated to real-world settings with caution until additional evidence is collected.
Our study had some shortcomings. First, the majority of ultrasonography was not performed by board-certified radiologists, so comparing our results with those of other researchers may yield some discrepancy. However, since our study objective was to pathologically investigate ultrasonographically “abnormal” gallbladders, and we histologically reclassified mucocele versus non-mucocele specimens, the results of our study should be regarded as reasonable enough to reach a conclusion. The second was a partial lack of clinical and clinicopathological information due to our retrospective approach. This impaired the completeness of evaluation on relationships among various gallbladder diseases and clinical/clinicopathological parameters. Thirdly, we could not speciate bacteria in the gallbladder lumen because we relied on cost-effective histochemistry to evaluate bacterial pathogens. Thinking of a large number of cases of positive bacterial detection (more than 60 cases in total), however, an attempt to speciate all the pathogens through molecular methodology (PCR or next-generation sequencing) was beyond our capacity. In the future, we or other investigators are encouraged to plan accordingly to take care of these matters.