Aim: This study was performed to evaluate the diagnostic value of hepatocyte-derived microRNA (miRNA)-122 in acute and chronic hepatitis of dogs.

Materials and Methods: A total of 26 dogs presented at Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Cairo University, 16 dogs out of 26 showing clinical signs of hepatic insufficiency were subjected to clinical, ultrasonographic, hematobiochemical and ultrasound-guided fine-needle biopsy for cytological and histopathological investigations. On the basis of these results, 7 dogs out of 16 dogs were found to be suffering from acute hepatitis and 9 dogs suffering from chronic hepatitis. 10 clinically healthy dogs were kept as control. Serum hepatocyte-derived miRNA-122 was analyzed by real-time quantitative polymerase chain reaction in all dogs.

Results: The dogs suffering from acute hepatitis manifested jaundice, vomiting, and depression while dogs with chronic hepatitis manifested anorexia, abdominal distension, weight loss, and melena. Hematological parameters showed normocytic normochromic anemia and thrombocytopenia in both acute and chronic hepatitis groups. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin were significantly higher than control values in acute hepatitis. In chronic hepatitis, total protein and albumin were significantly lower than control values with normal ALT, AST, ALP, and gamma-glutamyltransferase values. Ultrasonography revealed a diffuse decrease in hepatic echogenicity in acute hepatitis while the increase in hepatic echogenicity and anechoic ascetic fluid in chronic hepatitis. Cytology revealed hepatic vacuolar degeneration and histopathology revealed necrosis and apoptosis of hepatocyte in acute hepatitis while revealed massive fibrous tissue proliferation in hepatic parenchyma in chronic hepatitis. Serum miRNA-122 analysis, normalized for glyceraldehyde-3- phosphate dehydrogenase expression revealed a significant increase in acute hepatitis accompanied with elevation in ALT and AST, while in chronic hepatitis, elevation of serum miRNA-122 was accompanied with ALT and AST of the normal range.

Conclusion: Serum hepatocyte-derived miRNA-122 is of diagnostic value and highly stable blood indicator for the detection of hepatocellular injury in dogs than aminotransferases, especially in cases where aminotransferases do not exceed normal serum level.

Keywords: canine, cytology and histopathology, hepatitis, hepatocyte derived miRNA-122, ultrasonography.

1. Poldervaart, J.H., Favier, R.P., Penning, L.C., Van den Lngh, T.S. and Rothuizen, J. (2009) Primary hepatitis in dogs: A retrospective review. J. Vet. Intern. Med., 23(1): 72-80. [Crossref] [PubMed]

2. Meyer, H.P. and Rothuizen, J. (2013) History and physical examination. In: Washabau, R.J. and Day, M. J., editors. Canine and Feline Gastroenterology. 1st ed. Ch. 61(liver). Saunders, St. Louis, MO, USA. p849-958.

3. Richter, K.P. (2003) Diseases of the liver and hepatobiliary system. In: Tams, T.R., editor. Small Animal Gastroenterology. 2nd ed, Elsevier Science, Philadelphia. p286-352.

4. Lidbury, J.A. and Steiner, J.M. (2013) Liver-diagnostic evaluation. In: Washabau, R.J. and Day, M.J., editor. Canine and Feline Gastroenterology. 1st ed. Ch. 61(liver). Saunders, St. Louis, MO, USA.

5. Verzijl, T., Spee, B. and Fieten, H. (2014) Hepatocyte-Derived miR-122 as an Early Serum Biomarker of Hepatocellular Injury in Labrador Retrievers. Research Project in Veterinary Medicine. Faculty of Veterinary Medicine of the Utrecht University.

6. Li, S., Zhu, J., Fu, H., Wan, J., Hu, Z., Liu, S., Li, J., Tie, Y., Xing, R., Zhu, J., Sun, Z. and Zheng, X. (2012) Hepato-specific microRNA-122 facilitates accumulation of newly synthesized miRNA through regulating PRKRA. Nucleic Acids Res., 40: 884-891. [Crossref] [PubMed] [PMC]

7. Laterza, O.F., Lim, L., Garrett-Engele, P.W., Vlasakova, K., Muniappa, N., Tanaka, W.K., Johnson, J.M., Sina, J.F., Fare, T.L., Sistare, F.D. and Glaab, W.E. (2009) Plasma microRNAs as sensitive and specific biomarkers of tissue injury. Clin. Chem., 55: 1977-1983. [Crossref] [PubMed]

8. Wang, K., Zhang, S., Marzolf, B., Troisch, P., Brightman, A., Hu, Z., Hood, L.E. and Galas, D.J. (2009) Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc. Natl. Acad. Sci. USA., 106: 4402-4407. [Crossref] [PubMed] [PMC]

9. Zhang, Y., Jia, Y., Zheng, R., Guo, Y., Wang, Y., Guo, H., Fei, M. and Sun, S. (2010) Plasma microRNA-122 as a biomarker for viral-, alcohol-,and chemical-related hepatic diseases. Clin. Chem., 56: 1830-1838. [Crossref] [PubMed]

10. Van der Meer, A.J., Farid, W.R.R., Sonneveld, M.J., de Ruiter, P.E., Boonstra, A., van Vuurren, A.J., Verheij, J., Hansen, B.E., de Knegt, R.J. and van der Laan, L.J.W. (2013) Sensitive detection of hepatocellular injury in chronic hepatitis C patients with circulating hepatocyte-derived microRNA-122. J. Viral Hepat., 20: 158-166. [Crossref] [PubMed]

11. Krol, J., Loedige, L. and Filipowicz, W. (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat. Rev. Genet., 11: 597-610. [Crossref] [PubMed]

12. Dirksen, K., Verzijl, T., van den Lngh, T.S., Vernooij, J.C., Van der laan, L.J., Burgener, L.A., Spee, B. and Fieten, H. (2016) Hepatocyte-derived microRNAs as sensitive serum biomarkers of hepatocellular injury in Labrador retrievers. Vet. J., 211: 75-81. [Crossref] [PubMed]

13. Nyland, T.G., Larson, M.M. and Mattoon, J.S. (2015). Liver. In: Mattoon, J.S. and Nyland, T.G., editor. Small Animal Diagnostic Ultrasound. 3rd ed. Ch. 9. Elsevier Saunders, St. Louis, MO. p332-399. [Crossref]

14. van Steenbeek, F.G., Van den Bossche, L., Grinwis, G.C., Kummeling, A., van Gils, I.H., Koerkamp, M.J., van Leenen, D., Holstege, F.C., Penning, L.C., Rothuizen, J., Leegwater, P.A. and Spee, B. (2013). Aberrant gene expression in dogs with portosystemic shunts. PLoS One., 8: e57662. [Crossref] [PubMed] [PMC]

15. Yuan, J.S., Reed, A., Chen, F. and Stewart, C.N. (2006) Statistical analysis of real-time PCR data. BMC Bioinf., 7: 85. [Crossref] [PubMed] [PMC]

16. Kerwin, S.C. (1995) Hepatic aspiration and biopsy techniques. Vet. Clin. North Am. Small Anim. Pract., 25: 275-91. [Crossref]

17. Chen, Y. and Verfaillie, C.M. (2014) MicroRNAs: The fine modulators of liver development and function. Liver Int., 34: 976-990. [Crossref] [PubMed]

18. Batt, R.M. and Twedt, D.C. (1994) Canine gastrointestinal disease. In: Wills, J.M. and Simpson, K.W. editors. Waltham Book of Clinical Nutrition of the Dog and Cat. Pergamon Press, Oxford, UK. p235-258.

19. James, A.A. and Pillai, U. (2011) Ultrasonographic diagnosis of cirrhosis in a dog. J. Vet. Anim. Sci., 42: 64-65.

20. Gaschen, L. (2009) Update on hepatobiliary imaging. Vet. Clin. North Am. Small Anim. Pract., 39: 439-467. [Crossref] [PubMed]

21. Brockus, C.W. (2011) Erythrocytes. In: Duncan and Prasse's Veterinary Laboratory Medicine: Clinical Pathology. 5th ed. Iowa State Press, Ames.

22. Elhiblu1, M.A., Dua, K., Mohindroo, J., Mahajan, S.K., Sood, N.K. and Dhaliwal, P.S. (2015) Clinico-hemato-biochemical profile of dogs with liver cirrhosis. Vet. World, 8: 487-491.

23. Prins, M., Schellens, C.J., van Leeuwen, M.W., Rothuizen, J. and Teske, E. (2010) Coagulation disorders in dogs with hepatic disease. Vet. J., 185: 163-168. [Crossref] [PubMed]

24. Center, S.A. and Magne, M.L. (1991) Historical, physical examination and clinicopathological features of portosystemic vascular anomalies in the dog and cat. Semin. Vet. Med. Surg. (Small Anim), 5: 83.

25. Johnson, S.E. and Sherding, R.G. (1994) Diseases of the liver and biliary tract. In: Bichard, S.J. and Sherding, R.G. editors. Saunders Manual of Small Animal Practice. Saunders, Philadelphia. p723-724.

26. Bunch, S.E. (2003) Diagnostic tests for the hepatobiliary System. In: Nelson, R.W. and Couto, C.G. editors. Small Animal Internal Medicine. 3rd ed., Vol. 2. Mosby, St. Louis, Mo. p487.

27. Davies, D. (2016) Common inflammatory liver diseases in the dog (part 1). Vet. Ireland J. 6:556-60.

28. Cermelli, S., Ruggieri, A., Marrero, J.A., Loannou, G.N. and Beretta, L. (2011) Circulating microRNAs in patients with chronic hepatitis C and Non-alcoholic fatty liver disease. PLoS One, 6: e23937. [Crossref]

29. Lewis, P.J.S., Dear, J., Platt, V., Simpson, K.J., Craig, D.G., Antoine, D.J., French, N.S., Dhaun, N., Webb, D.J., Costello, E.M., Neoptolemos, J.P., Moggs, J., Goldring, C.E. and Park, B.K. (2011) Circulating microRNAs as potential markers of human drug-induced liver injury. Hepatology, 54: 1767-1776. [Crossref] [PubMed]

30. Dirksen, K., Verzijl, T., Grinwis, G.C., Favier, R.P., Penning, L.C., Burgener, I.A., van der Laan, L.J., Fieten, H. and Spee, B. (2016) Use of serum microRNAs as biomarker for hepatobiliary diseases in dogs. J. Vet. Intern. Med., 30: 1816-1823. [Crossref] [PubMed] [PMC]

31. Maxie, G.M. and Newman, S.J. (2007) Urinary system In: Jubb, Kennedy and Palmer's Pathology of Domestic Animals. 5th ed. Elsevier Saunders, Ontario. p425-522.

32. Rothuizen, J., Bunch, S., Charles, J., Cullen, J., Desmet, V., Szatmari, V., Twedt, D., van den Lngh, T., Winkle, T.V., and Washabau, R. (2006) Sampling and handling of liver tissue. In: WASA Liver Standardization Group, editors. WSAVA Standards for Clinical and Histological Diagnosis of Canine and Feline Liver Disease, Philadelphia, Elsevier. p5-14. [Crossref]

---