Liver Disease In Dogs Research Articles

Evaluation of Hepatocyte-derived MiRNA-122, and Insulin-like Growth Factor 1 for the Diagnosis of Liver Diseases in Dogs

Evaluation of Hepatocyte-derived MiRNA-122, and Insulin-like Growth Factor 1 for the Diagnosis of Liver Diseases in Dogs

Circulating microRNA detection using electrochemical biosensor for rapid diagnosis of liver disease in dogs

We report a new combination of short probe and target overhangs during hybridisation with a flow-based sample cycling setup for sensitive and specific detection of microRNA-122 biomarker. This setup was used towards liver injury diagnostics in dogs.

The comparative performance of a custom Canine NanoString® panel on FFPE and snap frozen liver biopsies

Formalin-Fixed Paraffin Embedded (FFPE) biopsies would provide a critical mass of cases to allow investigation of canine liver disease, however their use is often limited by challenges typically associated with transcriptomic analysis. This study evaluates the capability of NanoString® to measure the expression of a broad panel of genes in FFPE liver samples. RNA was isolated from matched histopathologically normal liver samples using FFPE (n = 6) and snap frozen in liquid nitrogen (n = 6) and measured using a custom NanoString® panel. Out of the 40 targets on the panel, 27 and 23 targets were above threshold for non-diseased snap frozen and FFPE tissue respectively. The binding density and total counts were significantly reduced in the FFPE samples relative to the snap frozen samples (p = 0.005, p = 0.01, respectively), confirming a reduction in sensitivity. The concordance between the snap frozen and FFPE samples was high, with correlations (R) ranging between 0.88 and 0.99 between the paired samples. An additional 14 immune-related targets, undetectable the non-diseased FFPE liver, were above threshold when the technique was applied to a series of diseased samples, further supporting their inclusion on this panel. This use of NanoString® based analysis opens up huge opportunity for retrospective evaluation of gene signatures in larger caseloads through harnessing the capacity of archived FFPE samples This information used alongside clinical and histological data will not only afford a way to explore disease etiopathogenesis, it may also offer insight into sub-types of liver disease in dogs, which cannot be discerned using more traditional diagnostic methods.

Antioxidant and Hepatoprotective Effect of a Nutritional Supplement with Silymarin Phytosome, Choline Chloride, l-Cystine, Artichoke, and Vitamin E in Dogs.

Phytotherapy has been promoted for the treatment of liver diseases in dogs. The interest in identifying the antioxidant/hepatoprotective potential of various plants is increasing. Every 10 days for 30 days, forty dogs were subjected to blood sampling and hepatic ultrasound assessment. Clinically healthy dogs (group A) and dogs with liver enzyme and ultrasound hepatic aspects and sizes outside the physiological range (group B) were divided into two subgroups. Every day for 30 days, one subgroup received Epacare pet + pasta® and the other received a placebo. Differences due to groups were observed in dROM, SHp, AST and LDH serum concentrations. The administration of Epacarepet + pasta® for 30 days had an influence on the serum concentration of dROMs, SHp, AST, ALT, ALP, LDH, and urea. The application of paired Student's t-test showed a decrease in the longitudinal and transverse liver axis size. In conclusion, feed supplementation with Epacare pet + pasta® had a beneficial effect on the antioxidant status and liver enzymes in animals with liver enzymatic alterations and in healthy dogs.

Hepatic Diseases in Canine and Feline: A Review

Dogs and cats are belonging to canine and feline family respectively. The liver is the largest gland in the body and is located in the cranial abdomen between 3rd and 4th ribs in dogs and cats. This review is mainly focused on: to understand the anatomy and physiology of liver, the liver diseases pathophysiology, to diagnose the liver diseases, managemental and ameliorative methods of liver diseases. The dual blood supply to the liver is hepatic artery and portal vein. The function of liver includes the regulation of digestion and metabolism, the synthesis of hormones and proteins, immune response and filtering of toxins from the blood stream. Any problem that affects the liver is liver disease. Inflammation of liver is hepatitis. Hepatitis caused by infectious, non-infectious, auto-immune and reactive. It can be acute and chronic. The most encountered liver diseases in dogs and cats are hepticlipidosis, cholangiohepatitis, portosystemicshunt, cholelithiasis, choledocholithiasis, cholecystitis, pneumobilia and hepatic neoplsia. The clinical symptoms of liver diseases include jaundice, hepatic encephalopathy, gastro intestinal disorders and nonspecific signs include polyuria/polydipisia. The liver disease diagnosed based on history, liver function tests, medical imaging. The latest imaging procedures are endoscopic retrograde cholagio pancreatography (ERCP) and computed tomography (CT). For confirmatory diagnosis liver biopsy and histopathological interpretation is required. Therefore, based on diagnosis appropriate treatment should be selected: bile stasis is treated urodeoxycholic acid (URDA), fluid therapy include sugar and salt solution for replacement fluid loss, gastrointestinal protectors include ranitidine, cimitidine and lactulose, albumin to treat hypoalbuminimia, antioxidant for scavenging free radicals include vitamin E, Selenium, S-adenosyl-L-methionine (SAMe), diuretic fursimid for treatment of ascites and supplementation low protein diets. This laparoscopic technique for removal of gallstones and endoscopic retrograde cholagiopancreatography (ERCP) to treat gall stones on bile ducts and to widen the slipped ducts and liver transplantation for cirrhotic dogs and cats. Early accurate diagnosis and managing any predisposing factors that affects the health of dogs and cats are important for controlling the liver diseases.

Resting and postprandial serum bile acid concentrations in dogs with liver disease.

BackgroundSerum bile acids (SBAs) are frequently measured in dogs. However, there is limited data comparing SBAs in different liver diseases diagnosed according to standardized histological criteria.ObjectivesTo compare resting and postprandial SBAs, and determine their sensitivity and specificity, for various liver diseases in dogs.AnimalsThree hundred and forty‐one client‐owned dogs with suspected liver disease that had a liver biopsy and SBAs measured.MethodsMulticenter retrospective study. Cases were classified according to standardized histological criteria. The sensitivity and specificity of resting and postprandial SBAs for the diagnosis of each liver disease, and all liver diseases combined, were calculated.ResultsThe median resting SBAs were highest in dogs with cirrhosis (98.8 μmol/L; range, 6‐135) and congenital circulatory anomalies (CCa; 79.45 μmol/L; 0.3‐705). The highest median postprandial concentrations were found in CCa (126 μmol/L; 0‐726) and chronic hepatitis (CH; 54.3 μmol/L; 0‐260). Using the cut‐off value of 10 μmol/L, the highest sensitivities of resting SBAs were recorded in dogs with CCa (87.5%; 95% confidence interval, 76.8‐94.4) and CH (81.1%; 71.5‐88.6). The sensitivities of postprandial SBAs were the highest in cholangitis (100%; 47.8‐100.0) and CCa (91.1%; 78.8‐97.5). The specificities of resting and postprandial SBAs for all diseases were 49.3% (37.6‐61.1) and 29.7% (15.9‐47.0), respectively.Conclusions and Clinical ImportancePostprandial SBAs are more sensitive but less specific than resting SBAs for the diagnosis of liver disease. There were dogs in all categories of liver disease with resting SBAs <10 and >90 μmol/L. Therefore, careful interpretation of both normal and elevated values is required.

Bidens pilosa L. (Asteraceae) cultivated in Brazil on acute liver disease in dogs

ABSTRACT Bidens pilosa L. is a medicinal plant popularly used for treatment of liver diseases. In this study, the dry extract of aerial parts of Bidens pilosa and Silymarin, a phytocomplex obtained from the Silybum marianum fruits and marketed as hepatoprotective, were tested in dogs experimentally acutely intoxicated with carbon tetrachloride. The liver activity was evaluated by hematological and biochemical profiles, and histological and ultrasound analyzes. It was observed that the lowest serum activities of ALT and serum concentrations of total bilirubin occurred in the groups treated with the dry extract of Bidens pilosa, while only decreased serum concentrations of total bilirubin occurred in the group treated with Silymarin. Best liver recovery was also observed for the dry extract of B. pilosa at a 400mg/Kg dose by ultrasonography. This study showed that the dry extract of Bidens pilosa acted more efficiently in the treatment of acute toxic hepatitis induced in dogs than Silymarin.

Integral Intoxication Indices in Liver Diseases in Dogs: Clinical Characteristics and Relevance

One of the indicators characterizing the severity of endogenous intoxication is considered the integral indices of intoxication, in which the indicators of the leukocyte formula are used. By changes in the leukocyte formula, taking into account other hematological parameters, one can judge the severity of the pathological process and the effectiveness of the therapy. As a result of the analysis of integral intoxication indices, it was found that on the arrival day, leukocyte intoxication index, which is used to assess the intoxication level, indicated the average degree of intoxication of animals with liver damage. On the 10th day of the study, the best result was observed in the 3rd group of animals. The value of leukocytal intoxication index in dogs of this group became 1.74±0.07, which indicates a decrease in the level of intoxication to normal. The Dashtayants nuclear index isalso used to assess the severity of intoxication of the animal. When comparing different treatment regimens, it was found that therapy using fresh frozen plasma allows to rid the body of intoxication and improve the condition of the animal.

Dietary supplements containing silymarin as a supportive factor in the treatment of canine hepatopathies

We review canine dietary supplements, in particular silymarin and its hepatoprotective properties. Dietary supplements should be considered complements to conventional drug treatments, helping to prevent health issues, such as nutritional deficiencies and organ dysfunctions. The market for veterinary supplements is growing rapidly. Pet owners are increasingly eager to provide their animals with the best care available. The rapidly growing market of supplements is an opportunity for both the industry and veterinarians, who can introduce new treatment protocols supported by dedicated dietary supplements. The liver is one organ that can be nutritionally supported in case of dysfunction. In dogs, hepatopathies may be primary or may be secondary to an underlying problem. Toxins, drugs, and neoplasia are reported among the causes of liver dysfunctions. There are several substances with proven liver-beneficial properties. One of these is silybin, a hepatoprotector extracted from Silybum marianum. Dietary supplementation with silybin seems a reasonable tool to support a core treatment for liver disease. Laboratory tests have additionally shown that silybin supplementation helps reduce liver damage. The literature indicates that silybin acts not only as an antioxidant, but also as an antifibrotic agent and a stimulator of cell regeneration. Due to its multidirectional action, it can effectively reduce liver damage, or help prevent it. Independent of the reason for its administration (prevention or therapy), it has been reported effective in laboratory animals, dogs, and humans. This was confirmed by an observational study performed at the University Center for Veterinary Medicine at Poznań University of Life Sciences. A total of 15 dogs with diagnosed liver conditions were supplemented with silybin for 30 days. Significant improvements were reported in the liver blood markers after the supplementation period. All things considered, silybin dietary supplementation is a noteworthy strategy in prevention and treatment of liver diseases in dogs.

Remarkable frequency of a history of liver disease in dogs fed homemade diets with buckwheat.

In our nutrition consultation service we observed liver disease in 2 dogs of one owner who was feeding buckwheat. This led to the hypothesis that buckwheat may cause problems. The present retrospective study in a German and a Russian nutrition consultation service was carried out to see whether there is an increased incidence of liver disease in dogs fed buckwheat. A retrospective study was carried out on the nutrition consultation cases of the Chair of Animal Nutrition and Dietetics, LMU Munich and a Russian nutrition consultant. All cases of dogs with buckwheat in their nutritional history were evaluated and compared with randomly selected dogs that had not been fed buckwheat from the same case set. Two German and 1 Russian buckwheat samples were compared (appearance, nutrient content, starch gelatinization, flavonoids, fagopyrin) as well as cooking methods. In the years 2007-2017, 34 cases of dogs fed buckwheat were identified in Germany and 57 in Russia. Eighty-five control cases in Germany and 48 in Russia were evaluated. In Germany, the incidence of liver disease in dogs fed buckwheat was 32 %, while that of the control group was 3.5 %. However, in Russia there was no significant difference between dogs fed buckwheat and control dogs. The appearance of the German and Russian buckwheat differed, with smaller seeds and more greenish colour in the German specimens while the Russian buckwheat presented larger and more brownish seeds. There was no difference in the analyses of the 3 buckwheat samples in crude nutrient and rutin content. Quercetin, quercitrin and fagopyrin were not detectable in all three samples. The degree of starch gelatinization in the Russian sample was higher than in the German. In Russia it is recommended to remove the reddish scum during boiling whereas this is rarely mentioned in Germany. German buckwheat may represent a risk in canine diets. With the difference remaining unclear, it is recommended to refrain from feeding buckwheat to dogs. In dogs fed homemade diets and suffering from liver disease, buckwheat should be considered in the nutrition history.

Ammonium tetrathiomolybdate treatment of copper-associated hepatopathy in dogs.

BackgroundCopper‐associated hepatopathy (CAH) is a common cause of liver disease in dogs. Although d‐penicillamine can be an effective treatment, some dogs fail treatment or develop adverse effects. Ammonium tetrathiomolybdate (TTM) has been used to treat pathologic copper accumulation in other species, but its therapeutic potential for CAH is unknown.ObjectivesTo investigate short‐term safety and efficacy of TTM for treatment of CAH.AnimalsTen dogs with CAH.MethodsProspective study. All dogs were treated with TTM PO for 6 weeks, and hepatic biopsies were performed after the treatment course. Dog experiencing initial decreases in hepatic copper concentrations ([Cu]H) received 6 additional weeks of TTM treatment and underwent 1 additional biopsy. Physical and laboratory examinations were performed every 2 weeks for study duration.ResultsEight of 10 dogs had decreases in [Cu]H. Compared to baseline (median, 1606 μg/g; range, 572‐5158 μg/g), [Cu]H were decreased at 6 weeks (1033 μg/g, 450‐2975 μg/g; P = .04) and 12 weeks (931 μg/g, 218‐1677 μg/g; P = .02). Hepatic molybdenum concentrations increased >50‐fold (P < 0.001). Changes in histologic scores and hematologic and biochemical test results were variable and not significantly different from baseline. One dog developed presumed immune‐mediated anemia and thrombocytopenia, but it was unclear if this was related to TTM administration.Conclusions and Clinical ImportanceResults suggest that TTM can effectively decrease [Cu]H in some dogs with CAH. Larger studies are needed to determine the overall safety and efficacy of TTM for treating CAH and how it compares with current treatments.

Sensitivity and specificity of microRNA-122 for liver disease in dogs.

BackgroundCurrent tests for diagnosing liver disease in dogs are sub‐optimal. MicroRNA‐122 (miR‐122) is a sensitive and specific biomarker of liver injury in humans and rodents. Circulating miR‐122 could have utility in identifying dogs with liver disease.ObjectiveEstablish the reference interval for miR‐122 in healthy dogs and determine performance in a range of dog breeds with liver disease and control animals with non‐liver disease.AnimalsStored serum from 120 healthy dogs, 100 dogs with non‐liver diseases, and 30 dogs with histologically confirmed liver disease was analyzed.MethodsRetrospective study. Medical records of dogs with liver disease, non‐liver disease and healthy dogs were reviewed. Serum miR‐122 concentrations were measured by PCR and compared with the characteristics of the dogs and their conventional clinical measurements.ResultsIn healthy dogs the 2.5th, 50th, and 97.5th quartiles of miR‐122 were 110 (90% CI 80‐114), 594 (505‐682), and 3312 (2925‐5144) copies/μL, respectively. There was no difference between healthy dogs and dogs with non‐liver disease (median ± IQR: healthy dogs 609 [327‐1014] copies/μL; non‐liver disease 607 [300‐1351] copies/μL). miR‐122 was higher in dogs with liver disease (11 332 [4418‐20 520] copies/μL, P < .001 compared to healthy dogs). miR‐122 identified dogs with liver disease with high accuracy (receiver operating characteristic area under curve for comparison with healthy dogs: 0.93 [95% CI 0.86‐0.99]). The upper limit of normal for healthy dogs (3312 copies/μL) had a sensitivity of 77% and specificity of 97% for identifying liver disease.Conclusion and Clinical ImportanceLiver disease can be sensitively and specifically diagnosed in dogs by measurement of miR‐122.

The effect of ammonia on canine polymorphonuclear cells.

Hyperammonaemia is a common complication of liver disease in dogs. High concentrations of ammonia can be detrimental to dogs with liver disease for several reasons, notably by causing hepatic encephalopathy (HE) which describes the wide range of neurological abnormalities ranging from altered behaviour to seizures that are well recognised complications in dogs with hepatic disorders. In human patients with liver disease, hyperammonaemia has also been linked to the development of other systemic complications such as dysregulation of the innate immune system. In contrast, the effects of hyperammonaemia on the canine innate immune system is currently unknown. The aim of this study was to investigate the effects of ammonia on the oxidative burst activity of canine polymorphonuclear cells in vitro. Blood obtained from healthy dogs (n = 8) was incubated with escalating concentrations of ammonia ranging from 0 to 250μM, and the percentage of cells experiencing an oxidative burst was evaluated using a commercial kit (Phagoburst™) and flow cytometry. The spontaneous oxidative burst was evaluated without stimulation and also following stimulation with E coli. The pH of the blood was also measured at the differing ammonia concentrations. There was an increase in the percentage of cells experiencing a spontaneous oxidative burst from ammonia concentrations of 125μM (p = <0.05) and above (p = <0.01), with a 4.9 fold increase at 200μM (p = < 0.001). In those cells stimulated with E coli, incubation with increasing ammonia concentrations did not result in a significant difference in oxidative burst from baseline (p = 0.953). There was no statistically significant difference between the pH of the blood at the various ammonia concentrations (p = 0.2) suggesting that the difference in spontaneous oxidative burst was due to the ammonia rather than simply a change in pH conditions. In summary, the spontaneous oxidative burst of neutrophils was significantly increased from baseline. This supports a potential role of ammonia in contributing to innate immune system dysfunction in dogs with liver disease, and may present a future therapeutic target.

Hepatic concentrations of copper and other metals in dogs with and without chronic hepatitis.

Defects in copper metabolism have been described in several dog breeds, and recently, it has been suggested that changes in other essential trace elements could be involved in the pathogenesis of hepatic disease. This study measured hepatic copper accumulation and its interactions with other essential trace and toxic metals in dogs diagnosed with chronic hepatitis. Liver samples of 20 chronic hepatitis and 20 healthy dogs were collected. Samples were acid digested, and essential metals (cobalt, copper, iron, manganese, molibdenum, selenium and zinc) and toxic metals (arsenic, cadmium, mercury and lead) were analysed by inductively-coupled plasma mass spectrometry. Copper concentrations were significantly higher in dogs affected by hepatic disease than in controls. Dogs having chronic hepatitis with liver copper concentration greater than 100 mg/kg wet weight showed statistically higher cobalt, manganese and zinc concentrations than dogs having chronic hepatitis with liver copper concentrations less than 100 mg/kg wet weight and controls. Toxic metal concentrations were low - in all cases below the threshold associated with toxicity in dogs. Dogs with chronic hepatitis not only have increased concentrations of copper in the liver but also increased concentrations of cobalt, manganese and zinc; measurement of these elements may perhaps aid in diagnosis of liver disease in dogs.

Sinusoidal endothelial cell and hepatic stellate cell phenotype correlates with stage of fibrosis in chronic liver disease in dogs.

We evaluated the extent of hepatic fibrosis in chronic liver disease of dogs using a modification of Ishak's staging criteria for human chronic liver disease, and examined the association of stage of fibrosis with immunophenotypic markers of transdifferentiation of hepatic sinusoidal endothelial cells and hepatic stellate cells. Formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained liver biopsy specimens from 45 case dogs with chronic liver disease and 55 healthy control dogs were scored for the presence and extent of fibrosis. This stage score for fibrosis strongly correlated with upregulated von Willebrand factor (vWF) expression in lobular sinusoidal endothelial cells (Spearman correlation coefficient [SCC] = 0.57, p < 0.05). Immunoreactivity for vWF factor was identified in 68.9% of case biopsies, varying in distribution from periportal to diffuse, whereas vWF immunoreactivity was identified in only 14.5% of control specimens, and was restricted to the immediate periportal sinusoids. The majority of both case and control biopsies exhibited similar prominent lobular perisinusoidal expression of alpha-smooth muscle actin (α-SMA). A minority of specimens (17.8% of case biopsies, 1.8% of control biopsies) exhibited low perisinoidal α-SMA expression, and there was a weak negative correlation between α-SMA expression and stage of fibrosis (SCC = -0.29, p = 0.0037). These results document a method for staging the severity of fibrosis in canine liver biopsies, and show a strong association between fibrosis and increased expression of vWF in hepatic sinusoidal endothelial cells.

Hepatocyte-derived microRNAs as sensitive serum biomarkers of hepatocellular injury in Labrador retrievers

Common parenchymal liver diseases in dogs include reactive hepatopathies and primary hepatitis (acute or chronic). In chronic hepatitis, there is usually a long subclinical phase. Specific clinical signs become overt only when liver damage is severe and in this phase, treatment is usually less effective. Limited data are available regarding the sensitivity of liver enzyme activity or biomarkers for early detection of subclinical hepatitis. Hepatocyte-derived microRNAs (HDmiRs) were recently identified as promising biomarkers for hepatocellular injury in multiple species. Here, the potential of the HDmiRs miR-122 and miR-148a as sensitive diagnostic biomarkers for hepatocellular injury in Labrador retrievers was investigated.Samples from 66 Labrador retrievers with histologically normal livers, high hepatic copper, and with various forms of liver injury were evaluated for serum alanine aminotransferase (ALT) activity and microRNA values. Median values of HDmiR-122 were 34.6 times higher in dogs with liver injury and high ALT than in normal dogs (95% confidence intervals [CI], 13–95; P < 0.001). HDmiR-122 values were significantly increased in dogs with liver injury and normal ALT (4.2 times; 95% CI, 2–12; P < 0.01) and in dogs with high hepatic copper concentrations and unremarkable histopathology (2.9 times; 95% CI, 1.1–8.0; P < 0.05). Logistic regression analyses demonstrated that miR-122 and miR-148a were both predictors of hepatocellular injury. The sensitivity of miR-122 was 84% (95% CI, 73–93%), making it superior to ALT (55%; 95% CI, 41–68%) for the detection of hepatocellular injury in Labrador retrievers (P < 0.001). This study demonstrated that serum HDmiR, particularly miR-122, is a highly sensitive marker for the detection of hepatocellular injury in Labrador retrievers and is a promising new biomarker that may be used for early detection of subclinical hepatitis in dogs.

Canine hepacivirus and idiopathic hepatitis in dogs from a Dutch cohort

Liver diseases are highly prevalent in the general dog population, though the etiology is often unknown. Recently a homolog of human hepatitis C virus was discovered in dogs with respiratory infections. Although this canine hepacivirus (CHV) was detectable in some liver samples, a clear link with liver disease has not been established. A recent study by Bexfield etal. showed that in a large cohort of dogs from the UK with idiopathic hepatitis, no evidence can be found for exposure to, or carrier state of CHV both in liver and in serum. The authors however state that 'the absence of CHV infection on dogs from the UK might not represent the global ecology of the virus'. We investigated CHV-infection in 267 liver biopsies from 120 dogs idiopathic hepatitis and 135 control animals, in a population from the Netherlands. Using a highly sensitive PCR assay for CHV-NS3, no CHV was detected in all 267 liver samples. Our data show that the lack of association between canine hepacivirus and chronic liver disease in dogs is not limited to the UK, but is also found in an independent cohort from the European continent.

Hepatopatia crônica associada a tratamento prolongado com fenobarbital em caninos

Chronic liver diseases are common in dogs, however, their causes are often undetermined. They have been associated with long-term phenobarbital therapy, used in large-scale as anticonvulsivant drug in small animal clinics. In the investigation of etiology for liver disease in dogs it’s important to check the prolonged use of phenobarbital. A retrospective study of canine necropsies performed at Laboratory of Veterinary Pathology of Universidade Federal do Rio Grande do Sul, was made from 2000 to 2010, and two cases of chronic diffuse hepatopathy in dogs under phenobarbital therapy (more than six months of treatment) were diagnosed. The main macroscopic findings, such as smaller, firmer and irregular surface of liver, ascites and other sites edema, and portosystemic shunts were observed. Histologically, the most important lesions were observed in the liver. There were diffuse fibrosis, bile ducts proliferation, mononuclear inflammatory infiltrate, cholestasis, and, in one case also occurred regenerating nodules of hepatocytes with vacuolization of hepatocytes cytoplasm. The diagnosis of chronic hepatopathy was based on these findings, and the intensity of the injuries was consistent with the time of treatment using phenobarbital.

Hepatic Injury Correlates With Apoptosis, Regeneration, and Nitric Oxide Synthase Expression in Canine Chronic Liver Disease

Assessment of the clinical severity, pathogenesis, and prognosis of canine chronic liver disease poses significant challenges to clinicians and pathologists, relating in part to a lack of standardized terminology and assessment methods and also to a lack of understanding of the pathogenesis of chronic liver disease in the dog. This study graded the severity of necroinflammatory activity in chronic liver disease in dogs using a modification of Ishak's grading scheme for human chronic liver disease and examined the association of grade score with hepatocellular apoptosis, regeneration, nitric oxide synthase isoform expression, copper and iron accumulation, and indicators of oxidative stress. Formalin-fixed, paraffin-embedded hematoxylin and eosin (HE)-stained liver biopsies from 45 dogs with chronic liver disease and 55 healthy control dogs were graded for various morphologic components of liver injury and response. The cumulative score for grade of necroinflammatory activity was strongly and significantly correlated with immunoreactive labels for hepatocellular proliferation (Ki-67); apoptosis (cleaved caspase-3); inducible nitric oxide synthase (iNOS) in lobular, portal, and septal stromal cells; endothelial nitric oxide synthase (eNOS) in hepatocytes and lobular, portal, and septal stromal cells; and total stainable hepatic iron. A weaker significant correlation was found between grade and accumulation of hepatocellular copper. No significant correlation was found between grade and immunoreactivity for malondialdehyde-protein adducts. These results document a method for grading of the severity of necroinflammatory disease in canine liver biopsies and show an association with increased iNOS and eNOS expression.

Correlation Between Hepatic Lipogranuloma Pigmentation and Stage of Cronic Liver Disease in Dogs

Introduction: Hepatic lipogranulomas are poorly defined lesions and have an undetermined significance and etiology. They are often found in the hepatic parenchyma of dogs, and are associated with previous hepatocyte death and circulatory disorders. Aims: The aim of the present study was to determine the correlation between the pigmentation and localization of hepatic lipogranulomas and the evolutive stage of chronic liver disease in dogs. Materials and methods: The study was conducted on a total of 25 liver samples obtained after necropsy from dogs with chronic liver disease (cirrhosis, lobular dissecting hepatitis, chronic hepatitis) with or without portosystemic shunts. Sections were stained with hematoxylin-eosin method for morphological diagnosis and for the identification of lipogranulomas. For characterization of intracytoplasmic pigments special histochemical methods were used (Perl’s reaction, PAS reaction, Oil red, Hall method, Rodamine stain). Immunohistochemical exam using antibodies against CD 68, and fluorescence microscopy was also performed. Results: In the present study we described two types of hepatic lipogranulomas: diffuse septal and intralobular well delimited. The grade of pigmentation and chemical composition of intracytoplasmic pigments changed over time, if in the initial phases lipogranulomas were diffuse, less pigmented and pigments were predominantly represented by ceroid, in later stages (with portosystemic shunts) lipogranulomas were heavily pigmented, pigments being represented primarily by hemosiderin. Conclusion: In the present study we showed that pigmentation of hepatic lipogranulomas is more intense in the late stages of chronic liver disease, when portal hypertension with portosystemic shunts occurs and the predominant pigment changes from ceroid to hemosiderin.