Liver histopathological findings and clinical associations in dogs undergoing cholecystectomy for gallbladder mucocele

Abstract

Gallbladder mucocele is frequently associated with concurrent hepatic pathology. However, studies examining the clinical relevance of concurrent liver histopathological findings in this condition remain scarce. This retrospective study evaluated liver histopathological findings in dogs that underwent cholecystectomy with concurrent liver biopsy and investigated their associations with preoperative variables, postoperative alanine aminotransferase (ALT) kinetics, and overall survival. Vacuolar degeneration was the most common histopathological finding, followed by hepatic fibrosis and neutrophilic cholangiohepatitis. The severity of neutrophilic cholangiohepatitis was positively correlated with preoperative serum bilirubin concentrations and common bile duct diameter and was associated with reduced postoperative survival. Conversely, the severity of fibrosis was primarily associated with the duration of postoperative ALT elevation. These findings suggest that concurrent liver histopathology, particularly the severity of neutrophilic cholangiohepatitis, may provide clinically relevant information regarding preoperative cholestatic changes and postoperative outcomes in dogs that underwent cholecystectomy for gallbladder mucocele.

INTRODUCTION

Gallbladder mucocele is a major biliary tract disease in dogs and may result in extrahepatic biliary obstruction, gallbladder rupture, bacterial infection, and systemic complications [3, 4]. Cholecystectomy remains the treatment of choice for dogs with gallbladder mucocele. Although historically high perioperative mortality has been reported [7], more recent studies have described improved outcomes after cholecystectomy, with one study reporting that 96% of dogs survived to discharge and 85% were alive at follow-up [5].

Previous studies have reported concurrent liver histopathological abnormalities, including cholangiohepatitis, fibrosis, and vacuolar degeneration, in dogs with gallbladder mucocele [2, 4]. However, the clinical significance of these lesions remains incompletely defined. In particular, relationships between lesion severity and preoperative cholestatic abnormalities have not been well characterized. Previous reports have suggested associations between preoperative findings and cholangitis/cholangiohepatitis as well as frequent concurrence of neutrophilic cholangitis/cholangiohepatitis with biliary disease or biliary obstruction [1, 2]; however, whether gallbladder disease or biliary obstruction contributes directly to the development of these inflammatory lesions remains unclear. Evaluation of serum bilirubin concentration and common bile duct diameter may help clarify these associations, and bacterial culture results may provide additional context when available.

Although short-term and long-term outcomes after cholecystectomy for gallbladder mucocele have been described, few studies have specifically examined the clinical relevance of concurrent liver histopathological findings [2, 4]. While postoperative biochemical improvement has been reported after cholecystectomy [5], persistent postoperative alanine aminotransferase (ALT) elevation is observed in some dogs and may complicate postoperative management. However, the relationship between postoperative ALT course and concurrent liver histopathological lesions remains unclear, especially in dogs with more advanced cholestatic change.

We hypothesized that the severity of concurrent liver histopathological lesions would be associated with more severe preoperative cholestatic change, prolonged postoperative ALT elevation, and shorter survival after cholecystectomy. The objectives of this study were to describe liver histopathological findings in dogs with gallbladder mucocele who had undergone cholecystectomy and to evaluate the associations of those findings with preoperative variables, postoperative ALT course, and survival.

MATERIALS AND METHODS

Medical records of dogs that underwent cholecystectomy with concurrent liver biopsy for gallbladder mucocele at a Minami Animal Hospital Group facility between October 2010 and March 2017 were retrospectively reviewed. Dogs were eligible for inclusion if they underwent cholecystectomy for gallbladder mucocele and had liver biopsy specimens available for histopathological evaluation. Gallbladder mucocele was provisionally diagnosed by ultrasonography and definitively diagnosed by gross findings in the excised gallbladder and histopathological examination. A total of 52 dogs met the inclusion criteria.

Recorded data included age, breed, clinicopathological findings, diagnostic imaging results, and outcome. Common bile duct diameter was evaluated as the absolute maximal diameter measured on preoperative CT images; adjustment for body size was not performed because of the retrospective nature of the study. Bacterial culture and antimicrobial susceptibility testing were performed when samples of bile or gallbladder contents, or both, had been collected at surgery as part of routine clinical management. When follow-up information was incomplete, the referring veterinarian or owner was contacted to obtain additional postoperative information, including serum ALT activity, duration of ALT elevation, the presence of liver-related disease, and, when applicable, cause of death.

Liver tissue samples from all cases were stored as formalin-fixed, paraffin-embedded blocks. Sections were stained with hematoxylin and eosin and special stains, including Azan and copper stains, and were re-evaluated by a veterinary pathologist. Histopathological evaluation included assessment of neutrophilic cholangiohepatitis, lymphocytic/plasmacytic cholangiohepatitis, granulomatous hepatitis, hepatocellular vacuolar degeneration, serositis, fibrosis, copper-associated changes, and bile thrombi. The severity of inflammatory lesions, fibrosis, and bile thrombi was graded as mild, moderate, or severe, as applicable.

Postoperative outcomes of interest included death within 14 days after surgery, duration of postoperative serum ALT activity above the reference upper limit (150 U/L), and survival time. For analyses of postoperative ALT activity, persistent postoperative ALT elevation was defined as serum ALT activity >150 U/L. The duration of postoperative ALT elevation was defined as the interval from surgery to the first documented decrease to ≤150 U/L. Survival time was defined as the interval from the date of cholecystectomy to death from any cause or last follow-up.

Ethical considerations

This study was a retrospective review of medical records and archived histopathological specimens obtained during routine clinical management. No animals underwent additional procedures for the purposes of this study. According to the policies of the participating institutions, formal ethical approval was not required for this type of retrospective clinical study.

Statistical analysis

Data normality was assessed using the Shapiro-Wilk test. Associations between histopathological severity scores and preoperative serum bilirubin concentration or common bile duct diameter were evaluated using Spearman rank correlation coefficients. Duration of postoperative ALT elevation was compared among histopathological severity groups using the Kruskal-Wallis test. When an overall group difference was detected, post-hoc pairwise comparisons were performed using the Steel-Dwass test. Associations between categorical variables, including bacterial culture results and death within 14 days after surgery, were assessed using Fisher’s exact test. Dogs alive at last follow-up or lost to follow-up were censored. Survival distributions among groups based on liver histopathological findings were estimated using the Kaplan-Meier method and compared using the log-rank test.

All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). A value of P<0.05 was considered to indicate significant results.

RESULTS

Clinical characteristics

Fifty-two dogs were included. Median age was 10 years (range: 3–17 years), median body weight was 4.3 kg (range: 2.0–18.5 kg), and the most common breeds were Miniature Dachshund, Chihuahua, and Toy Poodle.

Median preoperative serum bilirubin concentration was 1.35 mg/dL (range: 0.1–10.2 mg/dL), and values were above the reference interval in 32 of 52 dogs (61%). Common bile duct diameter was measured by preoperative CT in 33 of 52 dogs, and dilation ≥4 mm was identified in 19 of these 33 dogs (58%). Bacterial culture and antimicrobial susceptibility testing were performed in 43 of 52 dogs, and the results were positive for 16 of 43 dogs (37%).

Median follow-up time was 942 days (range: 2–3,336 days). Four of 52 dogs (7.6%) died within 14 days after surgery. Causes of death included disseminated intravascular coagulation secondary to bacterial peritonitis with postoperative biliary leakage (n=2), disseminated intravascular coagulation (n=1), and exacerbation of hepatic lymphoma (n=1). Overall, 28 of 52 dogs died during the study period, and median postoperative survival time was 1,296 days (range: 2–2,990 days). Median postoperative serum ALT activity was 417 U/L, and median duration of postoperative ALT elevation was 18 days (range: 0–850 days).

Liver histopathological findings

Histopathological evaluation revealed neutrophilic cholangiohepatitis in 37 of 52 dogs (71%), including 28 mild, 8 moderate, and 1 severe cases. Hepatocellular vacuolar degeneration was present in 42 of 52 dogs (80%), including 30 mild, 10 moderate, and 2 severe cases. Fibrosis was observed in 42 of 52 dogs (80%), including 28 mild and 14 moderate cases. Representative histopathological findings observed in dogs with neutrophilic cholangiohepatitis in the present cohort included multifocal infiltration and expansion of portal areas and adjacent hepatic parenchyma by variable numbers of inflammatory cells, predominantly composed of neutrophils and fewer lymphocytes and/or plasma cells. The inflammatory neutrophils often separated and surrounded bile ducts and occasionally infiltrated into biliary epithelium and surrounding hepatic parenchyma. Adjacent hepatocytes were often swollen with vacuolated, lacy cytoplasm (vacuolar degeneration) with occasional centrilobular, cytoplasmic yellow pigment (lipofuscin). Biliary epithelium was often vacuolated. There were increased numbers of small bile ducts surrounded by variable numbers of fibroblasts (fibrosis) (Fig. 1). In addition, scattered aggregates of Kupffer cells containing variable amounts of granular to globular, yellow to brown, cytoplasmic pigment (hemosiderin or lipofuscin) were also noted. One dog each had lymphocytic/plasmacytic cholagiohepatitis and granulomatous hepatitis, both classified as moderate cases. Copper-associated changes were suspected in six dogs based on centrilobular copper accumulation, although quantitative copper analysis was not performed. Lymphoma was identified in one dog.

Fig. 1.

Representative photomicrographs of moderate neutrophilic cholangiohepatitis. Moderate periportal inflammatory cell infiltration, predominantly of neutrophils, is visible in hematoxylin and eosin staining images (A), with moderate fibrosis in Azan staining images (B). Bar=100 µm.

Associations between preoperative variables and histopathological findings

Among dogs with both bacterial culture results and evaluable neutrophilic cholangiohepatitis classification, bacterial culture positivity did not differ significantly between dogs without neutrophilic cholangiohepatitis and those with neutrophilic cholangiohepatitis (1/8 [12.5%] vs 13/32 [40.6%]; P=0.222), and no significant associations were identified between culture results and other histopathological findings. Neutrophilic cholangiohepatitis severity was significantly positively correlated with preoperative serum bilirubin concentration (Spearman’s rho=0.468, P=0.0012) and common bile duct diameter (Spearman’s rho=0.473, P=0.0126) (Fig. 2). Median preoperative serum bilirubin concentrations were 0.3 mg/dL [0.2–0.45] in dogs without neutrophilic cholangiohepatitis (n=10), 0.9 mg/dL [0.4–4.675] in dogs with mild lesions (n=26), 3.0 mg/dL [1.95–4.875] in dogs with moderate lesions (n=8), and 7.8 mg/dL in the single dog with severe lesions (n=1). Median common bile duct diameters were 2.7 mm [2.0–3.25] in dogs without neutrophilic cholangiohepatitis (n=8), 5.2 mm [3.1–6.1] in dogs with mild lesions (n=17), 5.55 mm [4.575–6.675] in dogs with moderate lesions (n=4), and 7.0 mm in the single dog with severe lesions (n=1). No significant correlations were identified between other histopathological findings and these preoperative variables.

Fig. 2.

Association between neutrophilic cholangiohepatitis severity and preoperative serum bilirubin concentration (A) and common bile duct diameter (B) shown as box-and-whisker plots.

Survival and histopathological findings

No significant associations were observed between liver histopathological findings and death within 14 days after surgery. Survival differed significantly among neutrophilic cholangiohepatitis severity groups (log-rank P=0.0299) (Fig. 3). Median survival times were 2,187 days for dogs without neutrophilic cholangiohepatitis (n=12), 2,126 days for dogs with mild lesions (n=28), and 1,022 days for dogs with moderate lesions (n=8); median survival time was shortest in dogs with moderate lesions. The single dog with severe neutrophilic cholangiohepatitis was included in the survival analysis and died 2,364 days after surgery, but this category was not interpreted separately because of the very small sample size. For fibrosis, median survival times were 1,455 days for dogs without fibrosis (n=10), 2,187 days for dogs with mild fibrosis (n=28), and 1,478 days for dogs with moderate fibrosis (n=14), with no significant differences among groups (log-rank P=0.446). No significant survival differences were identified for other histopathological findings.

Fig. 3.

Kaplan–Meier survival curves for dogs with gallbladder mucocele stratified by neutrophilic cholangiohepatitis severity after cholecystectomy.

Duration of postoperative serum ALT activity above 150 U/L and histopathological findings

No significant overall differences in the duration of postoperative ALT elevation were observed among neutrophilic cholangiohepatitis severity groups (P=0.602). Median durations were 13.5 days [5.5–52.5] in dogs without neutrophilic cholangiohepatitis (n=10), 15 days [4.0–76.5] in dogs with mild lesions (n=28), 151 days [40.0–324.5] in dogs with moderate lesions (n=7), and 6 days in the single dog with severe lesions (n=1); therefore, this value should be interpreted descriptively. Overall, the median duration was longest in dogs with moderate lesions. In contrast, duration of postoperative ALT elevation significantly differed among fibrosis severity groups (P=0.01). Median durations were 64 days [10–245] in dogs without fibrosis (n=9), 8 days [3.25–34] in dogs with mild fibrosis (n=26), and 54.5 days [12.5–330.25] in dogs with moderate fibrosis (n=14). A post-hoc pairwise comparison demonstrated a significant difference between dogs with mild fibrosis and those with moderate fibrosis. No significant differences were identified for other histopathological findings.

DISCUSSION

The most common liver histopathological findings in this cohort were hepatocellular vacuolar degeneration (80%), fibrosis (80%), and neutrophilic cholangiohepatitis (71%). Previous studies have also described fibrosis, vacuolar degeneration, and cholangiohepatitis as common concurrent lesions in dogs with gallbladder mucocele, although reported frequencies varied [2, 4]. In the study by Malek et al. [4], fibrosis, vacuolar degeneration, and cholangiohepatitis were reported in 21.6%, 2.7%, and 29.7% of dogs, respectively, whereas Jablonski et al. [2] reported corresponding frequencies of 73%, 42%, and 25%. The higher frequency of fibrosis in the present study and in the study by Jablonski et al. [2] than in the study by Malek et al. [4] may partly reflect the use of special stains for fibrosis detection. The higher proportion of neutrophilic cholangiohepatitis in the present cohort may also reflect differences in case characteristics. Positive bacterial culture results were more frequent in the present study (37%) than in the study by Malek et al. [4] (2%) or the study by Jablonski et al. [2] (12%), which may have contributed to this difference.

Neutrophilic cholangiohepatitis severity was positively correlated with preoperative serum bilirubin concentration and common bile duct diameter measured by CT. As hyperbilirubinemia and common bile duct dilation are clinically important indicators of cholestatic change or biliary obstruction, these findings suggest an association between neutrophilic cholangiohepatitis and cholestatic or obstructive hepatobiliary changes. Histopathological classifications of canine and feline liver disease describe extrahepatic cholestasis as being associated with portal edema, neutrophilic portal inflammation, bile duct epithelial changes, biliary proliferation, and, in chronic cases, fibrosis and mixed inflammatory infiltrates [6]. Harrison et al. [1] reported that canine cholangitis and cholangiohepatitis were most commonly associated with biliary disease and biliary obstruction. In previous studies, hyperbilirubinemia and common bile duct dilation were reported less frequently than in the present cohort, and the proportion of dogs with cholangiohepatitis was also lower [2, 4]. Taken together, these findings suggest that more marked cholestatic or obstructive hepatobiliary changes in the present cohort may have contributed to the higher proportion and greater severity of neutrophilic cholangiohepatitis. However, these findings should be interpreted cautiously because neutrophilic cholangiohepatitis may also occur independently of biliary obstruction, the sample size was limited, and the retrospective observational design precludes determination of whether these associations indicate a direct causal relationship or concurrent manifestations of more advanced hepatobiliary disease.

Prolonged postoperative ALT elevation was observed in some dogs after cholecystectomy. Although no significant overall differences were observed in the duration of postoperative ALT elevation among neutrophilic cholangiohepatitis severity groups, the longest median duration was observed in dogs with moderate lesions. In contrast, postoperative ALT duration differed significantly among fibrosis severity groups, with the shortest duration observed in dogs with mild fibrosis and a significant difference between mild and moderate fibrosis. These findings suggest that concurrent hepatobiliary lesions may influence postoperative ALT course in some dogs. However, prolonged ALT elevation was also observed in some dogs without fibrosis or with only mild neutrophilic cholangiohepatitis, indicating that factors other than the histopathological lesions evaluated in this study may also have contributed.

Survival differed significantly among neutrophilic cholangiohepatitis severity groups, and median survival time was shortest in dogs with moderate lesions. This finding suggests that more severe neutrophilic inflammatory hepatobiliary change may be associated with poorer long-term outcome after cholecystectomy. Neutrophilic cholangiohepatitis severity was also positively correlated with preoperative serum bilirubin concentration and common bile duct diameter. Taken together, these findings suggest that neutrophilic cholangiohepatitis may be a clinically relevant histopathological marker associated with more advanced hepatobiliary disease and shorter postoperative survival. However, it remains unclear whether neutrophilic cholangiohepatitis itself contributed independently to reduced survival or whether it reflected more advanced biliary obstruction or cholestatic hepatobiliary disease. Because multivariable survival analysis, such as Cox proportional hazards analysis, was not performed, the independent prognostic significance of neutrophilic cholangiohepatitis could not be determined in the present study. In contrast to the study by Jablonski et al. [2], which identified an association between fibrosis and shorter short- and mid-term survival, the present study did not identify a relationship between fibrosis and long-term survival. Differences in case composition, follow-up duration, and lesion distribution may partly explain these discrepancies. Given the retrospective design, lack of standardized postoperative management, and absence of multivariable survival analysis, these findings regarding survival should be interpreted cautiously.

Overall, these findings partially supported our hypothesis: greater severity of neutrophilic cholangiohepatitis was associated with more severe preoperative cholestatic change and shorter survival, whereas fibrosis severity was associated with postoperative ALT course rather than survival.

This study had some limitations. Detailed information regarding biopsy location and the number of biopsy sites was unavailable, precluding assessment of sampling variability. As this was a retrospective study conducted across multiple facilities over several years, perioperative and postoperative treatment protocols were not standardized. Variability in postoperative ALT testing intervals may have affected estimation of ALT duration. In addition, the timing of surgery, duration of clinical signs, degree and duration of preoperative stabilization, presence or absence of gallbladder rupture, progression of biliary obstruction, and time from diagnosis to intervention were not systematically evaluated. Therefore, this study cannot determine whether earlier surgical intervention would reduce the severity of hepatobiliary lesions or improve prognosis. Furthermore, because multivariable survival analysis was not performed, potential confounding among neutrophilic cholangiohepatitis severity, hyperbilirubinemia, common bile duct dilation, and survival could not be fully assessed. Cause-of-death information was obtained largely from owner reports, which may have limited accurate classification, particularly in this older population. Finally, the overall sample size and small size of some histopathological subgroups limited statistical power. Because long-term survival was one of the outcomes of interest, inclusion of past cases was necessary to allow sufficient follow-up, which may limit direct generalization to current patient populations. In conclusion, liver histopathological findings in dogs with gallbladder mucocele were associated with clinically relevant preoperative and postoperative variables. Neutrophilic cholangiohepatitis severity was associated with preoperative cholestatic abnormalities and shorter postoperative survival, whereas fibrosis severity was associated with postoperative ALT course. These findings suggest that concurrent liver histopathological evaluation may provide clinically relevant information regarding preoperative cholestatic abnormalities, postoperative ALT course, and survival in dogs undergoing cholecystectomy for gallbladder mucocele.

Conflicts of Interest

The authors declare no conflicts of interest.