The liver-brain axis under the influence of chronic Opisthorchis felineus infection combined with prolonged alcoholization

   Our purpose was to model a combination of a prolonged consumption of ethanol with Opisthorchis felineus infection in mice. Four groups of C57BL/6 mice were compiled: OF, mice infected with O. felineus for 6 months; Eth, mice consuming 20 % ethanol; Eth+OF, mice subjected to both adverse factors; and CON, control mice not exposed to these factors. In the experimental mice, especially in Eth+OF, each treatment caused well-pronounced periductal and cholangio fibrosis, proliferation of bile ducts, and enlargement of areas of inflammatory infiltration in the liver parenchyma. Simultaneously with liver disintegration, the infectious factor caused – in the frontal cerebral cortex – the growth of pericellular edema (OF mice), which was attenuated by the administration of ethanol (Eth+OF mice). Changes in the levels of some proteins (Iba1, IL-1β, IL-6, and TNF) and in mRNA expression of genes Aif1, Il1b, Il6, and Tnf were found in the hippocampus and especially in the frontal cortex, implying region-specific neuroinflammation. Behavioral testing of mice showed that ethanol consumption influenced the behavior of Eth and Eth+OF mice in the forced swimming test and their startle reflex. In the open field test, more pronounced changes were observed in OF mice. In mice of all three experimental groups, especially in OF mice, a disturbance in the sense of smell was detected (fresh peppermint leaves). The results may reflect an abnormality of regulatory mechanisms of the central nervous system as a consequence of systemic inflammation under the combined action of prolonged alcohol consumption and helminth infection.

1. Adeva M.M., Souto G., Donapetry C., Portals M., Rodriguez A., Lamas D. Brain edema in diseases of different etiology. Neurochem Int. 2012;61(2):166-174. doi: 10.1016/j.neuint.2012.05.007

2. Akhmedov V.A., Kritevich M.A. Chronic opisthorchiasis is a multipleorgan pathology. Vestnik NGU. Seriya: Biologiya, Klinicheskaya Meditsina = Vestnik NSU. Series: Biology and Clinical Medicine. 2009;7(1):118-121. [https://nsu.ru/xmlui/bitstream/handle/nsu/4344/21.pdf] (in Russian)

3. Avgustinovich D.F., Marenina M.K., Zhanaeva S.Y., Tenditnik M.V., Katokhin A.V., Pavlov K.S., Sivkov A.Y., Vishnivetskaya G.B., Lvova M.N., Tolstikova T.G., Mordvinov V.A. Combined effects of social stress and liver fluke infection in a mouse model. Brain Behav Immun. 2016;53:262-272. doi: 10.1016/j.bbi.2016.01.012

4. Avgustinovich D., Kovner A., Kashina E., Shatskaya N., Vishnivetskaya G., Bondar N., Lvova M. The pathogenic potential of the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice. Int J Parasit. 2021;51:353-363. doi: 10.1016/j.ijpara.2020.10.003

5. Avgustinovich D., Kizimenko A., Marenina M., Lvova M., Kovner A., Orlovskaya I., Toporkova L., Goiman E., Tsyganov M., Ponomarev D. Prolonged liver fluke infection combined with alcoholization: an experimental mouse model. Exp Parasitol. 2022a;242: 108399. doi: 10.1016/j.exppara.2022.108399

6. Avgustinovich D.F., Tenditnik M.V., Bondar N.P., Marenina M.K., Zhanaeva S.Y., Lvova M.N., Katokhin A.V., Pavlov K.S., Evseenko V.I., Tolstikova T.G. Behavioral effects and inflammatory markers in the brain and periphery after repeated social defeat stress burdened by Opisthorchis felineus infection in mice. Physiol Behav. 2022b;252:113846. doi: 10.1016/j.physbeh.2022.113846

7. Bajaj J.S. Alcohol, liver disease and the gut microbiota. Nat Rev Gastroenterol Hepatol. 2019;16(4):235-246. doi: 10.1038/s41575-018-0099-1

8. Belzung C. Hippocampal mossy fibres: implication in novelty reactions or in anxiety behaviours? Behav Brain Res. 1992;51(2):149-155. doi: 10.1016/s0166-4328(05)80208-6

9. Bernstein D.E., Dickinson G.M., Kim K.-J., Al Karawi M.A., Barkin J.S. Parasitic causes of pancreatic and biliary tract disease: a growing concern in a highly mobile population. Mil Med. 1994; 159(4):331-338. doi: 10.1093/milmed/159.4.331

10. Bilzer M., Roggel F., Gerbes A.L. Role of Kupffer cells in host defense and liver disease. Liver Int. 2006;26(10):1175-1186. doi: 10.1111/j.1478-3231.2006.01342.x

11. Bishehsari F., Magno E., Swanson G., Desai V., Voigt R.M., Forsyth C.B., Keshavarzian A. Alcohol and gut-derived inflammation. Alcohol Res. 2017;38(2):163-171

12. Blednov Y.A., Bajo M., Roberts A.J., Da Costa A.J., Black M., Edmunds S., Mayfield J., Roberto M., Homanics G.E., Lasek A.W., Hitzemann R.J., Harris R.A. Scn4b regulates the hypnotic effects of ethanol and other sedative drugs. Genes Brain Behav. 2019;18(6): e12562. doi: 10.1111/gbb.12562

13. Boonpucknavig S., Boonpucknavig V., Tanvanich S., Doungchawee G., Thamavit W. Development of immune-complex glomerulonephritis and amyloidosis in Syrian golden hamsters infected with Opisthorchis viverrini. J Med Assoc Thai. 1992;75(Suppl.1):7-19

14. Brown R.E., Willner J.A. Establishing an “affective scale” for odor preferences of infant rats. Behav Neural Biol. 1983;38(2):251-260. doi: 10.1016/s0163-1047(83)90254-6

15. Butterworth R.F. Role of circulating neurotoxins in the pathogenesis of hepatic encephalopathy: potential for improvement following their removal by liver assist devices. Liver Int. 2003;23(Suppl.3):5-9. doi: 10.1034/j.1478-3231.23.s.3.1.x

16. Bychkov V.G., Solov’eva O.G., Khadieva E.D., Ivanova L.A., Kulikova S.V., Garchuk I.V., Orlov S.A., Sabirov A.Kh., Shilin K.O., Beliaeva M.I. Morphogenesis of the structural changes of the internal organs in superinvasive opisthorchiasis. Morfologiia = Morphology. 2011;140(5):22-27 (in Russian)

17. Chavarria L., Alonso J., Rovira A., Córdoba J. Neuroimaging in acute liver failure. Neurochem Int. 2011;59(8):1175-1180. doi: 10.1016/j.neuint.2011.09.003

18. Chen W., Chen S., Kang W.Y., Li B., Xu Z.M., Xiao Q., Liu J., Wang Y., Wang G., Chen S.D. Application of odor identification test in Parkinson’s disease in China: a matched case-control study. J Neurol Sci. 2012;316(1-2):47-50. doi: 10.1016/j.jns.2012.01.033

19. Choleris E., Thomas A.W., Kavaliers M., Prato F.S. A detailed ethological analysis of the mouse open field test: effects of diazepam, chlordiazepoxide and an extremely low frequency pulsed magnetic field. Neurosci Biobeh Rev. 2001;25(3):235-260. doi: 10.1016/s0149-7634(01)00011-2

20. Collins M.A., Neafsey E.J. Neuroinflammatory pathways in binge alcohol-induced neuronal degeneration: oxidative stress cascade involving aquaporin, brain edema, and phospholipase A2 activation. Neurotox Res. 2012;21:70-78. doi: 10.1007/s12640-011-9276-5

21. Collins M.A., Zou J.-Y., Neafsey E.J. Brain damage due to episodic alcohol exposure in vivo and in vitro: furosemide neuroprotection implicates edema-based mechanism. FASEB J. 1998;12(2):221-230. doi: 10.1096/fasebj.12.2.221

22. de la Monte S.M., Kril J.J. Human alcohol-related neuropathology. Acta Neuropathol. 2014;127(1):71-90. doi: 10.1007/s00401-013-1233-3

23. D’Mello C., Swain M.G. Liver-brain inflammation axis. Am J Physiol Gastrointest Liver Physiol. 2011;301(5):G749-G761. doi: 10.1152/ajpgi.00184.2011

24. Fernandez-Lizarbe S., Montesinos J., Guerri C. Ethanol induces TLR4/TLR2 association, triggering an inflammatory response in microglial cells. J Neurochem. 2013;126(2):261-273. doi: 10.1111/jnc.12276

25. Fowler A.K., Thompson J., Chen L., Dagda M., Dertien J., Dossou K.S., Moaddel R., Bergeson S.E., Kruman I.I. Differential sensitivity of prefrontal cortex and hippocampus to alcohol-induced toxicity. PLoS One. 2014;9(9):e106945. doi: 10.1371/journal.pone.0106945

26. Friard O., Gamba M. BORIS: a free, versatile open-source event-logging software for video/audio coding and live observations. Methods Ecol Evol. 2016;7:1325-1330. doi: 10.1111/2041-210X.12584

27. Fu Y., Yang M.-S., Jiang J., Ganesh T., Joe E., Dingledine R. EP2 receptor signaling regulates microglia death. Mol Pharmacol. 2015; 88(1):161-170. doi: 10.1124/mol.115.098202

28. Geil C.R., Hayes D.M., McClain J.A., Liput D.J., Marshall S.A., Chen K.Y., Nixon K. Alcohol and adult hippocampal neurogenesis: promiscuous drug, wanton effects. Prog Neuropsychopharmacol Biol Psychiatry. 2014;54:103-113. doi: 10.1016/j.pnpbp.2014.05.003

29. Gundamaraju R., Vemuri R.C. Opisthorchis viverrini (liver fluke) as the lot of baleful parasite of tropical region - A replete synopsis. Asian Pac J Trop Dis. 2014;4(1):61-66. doi: 10.1016/S2222-1808(14)60316-9

30. Harper C. The neuropathology of alcohol-specific brain damage, or does alcohol damage the brain? J Neuropathol Exp Neurol. 1998; 57(2):101-110. doi: 10.1097/00005072-199802000-00001

31. Harper C. The neuropathology of alcohol-related brain damage. Alcohol Alcohol. 2009;44(2):136-140. doi: 10.1093/alcalc/agn102

32. He J., Crews F.T. Increased MCP-1 and microglia in various regions of the human alcoholic brain. Exp Neurol. 2008;210(2):349-358. doi: 10.1016/j.expneurol.2007.11.017

33. Henderson N.D., Turri M.G., DeFries J.C., Flint J. QTL analysis of multiple behavioral measures of anxiety in mice. Behav Genet. 2004;34(3):267-293. doi: 10.1023/B:BEGE.0000017872.25069.44

34. Henriques J.F., Portugal C.C., Canedo T., Relvas J.B., Summavielle T., Socodato R. Microglia and alcohol meet at the crossroads: microglia as critical modulators of alcohol neurotoxicity. Toxicol Lett. 2018;283:21-31. doi: 10.1016/j.toxlet.2017.11.002

35. Hoogland I.C., Houbolt C., van Westerloo D.J., van Gool W.A., van de Beek D. Systemic inflammation and microglial activation : systematic review of animal experiments. J Neuroinflammation. 2015; 12:114. doi: 10.1186/s12974-015-0332-6

36. Huber V.J., Tsujita M., Yamazaki M., Sakimura K., Nakada T. Identification of arylsulfonamides as Aquaporin 4 inhibitors. Bioorg Med Chem Lett. 2007;17(5):1270-1273. doi: 10.1016/j.bmcl.2006.12.010

37. Jayakumar A.R., Tong X.Y., Ospel J., Norenberg M.D. Role of cerebral endothelial cells in the astrocyte swelling and brain edema associated with acute hepatic encephalopathy. Neuroscience. 2012;218: 305-316. doi: 10.1016/j.neuroscience.2012.05.006

38. Ketpueak T., Thiennimitr P., Apaijai N., Chattipakorn S.C., Chattipakorn N. Association of chronic opisthorchis infestation and microbiota alteration on tumorigenesis in cholangiocarcinoma. Clin Transl Gastroenterol. 2020;12(1):e00292. doi: 10.14309/ctg.0000000000000292

39. Khan M.I., Nikoui V., Naveed A., Mumtaz F., Zaman H., Haider A., Aman W., Wahab A., Khan S.N., Ullah N., Dehpour A.R. Antidepressant-like effect of ethanol in mice forced swimming test is mediated via inhibition of NMDA/nitric oxide/cGMP signaling pathway. Alcohol. 2021;92:53-63. doi: 10.1016/j.alcohol.2021.01.005

40. Kim B.Y., Park J.Y., Kim E.J., Kim B.G., Kim S.W., Kim S.W. The neuroplastic effect of olfactory training to the recovery of olfactory system in mouse model. Int Forum Allergy Rhinol. 2019;9(7):715-723. doi: 10.1002/alr.22320

41. Korbo L. Glial cell loss in the hippocampus of alcoholics. Alcohol Clin Exp Res. 1999;23(1):164-168. doi: 10.1111/j.1530-0277.1999.tb04039.x

42. Lowe P.P., Morel C., Ambade A., Iracheta-Vellve A., Kwiatkowski E., Satishchandran A., Furi I., Cho Y., Gyongyosi B., Catalano D., Lefeb vre E., Fischer L., Seyedkazemi S., Schafer D.P., Szabo G. Chronic alcohol-induced neuroinflammation involves CCR2/5-dependent peripheral macrophage infiltration and microglia alterations. J Neuroinflammation. 2020;17(1):296. doi: 10.1186/s12974-020-01972-5

43. Lucki I. A prescription to resist proscriptions for murine models of depression. Psychopharmacology (Berl). 2001;153(3):395-398. doi: 10.1007/s002130000561

44. Lvova M., Zhukova M., Kiseleva E., Mayboroda O., Hensbergen P., Kizilova E., Ogienko A., Besprozvannykh V., Sripa B., Katokhin A., Mordvinov V. Hemozoin is a product of heme detoxification in the gut of the most medically important species of the family Opisthorchiidae. Int J Parasitol. 2016;46(3):147-156. doi: 10.1016/j.ijpara.2015.12.003

45. Lvova M.N., Shevelev O.B., Serdobintseva V.V., Kalinin D.V., Starostenko D.A., Zavjalov E.L., Krivoshapkin A.L., Logachev P.V., Mordvinov V.A., Avgustinovich D.F. Effect of silicon dioxide nanoparticles on Syrian hamsters infected by Opisthorchis felineus: 1H MRS study of the brain. Dokl Biochem Biophys. 2020;495(1): 319-324. doi: 10.1134/S1607672920060095

46. Lvova M.N., Ponomarev D.V., Tarasenko A.A., Kovner A.V., Minkova G.A., Tsyganov M.A., Li M., Lou Y., Evseenko V.I., Dushkin A.V., Sorokina I.V., Tolstikova T.G., Mordvinov V.A., Avgustinovich D.F. Curcumin and its supramolecular complex with disodium glycyrrhizinate as potential drugs for the liver fluke infection caused by Opisthorchis felineus. Pathogens. 2023;12(6):819. doi: 10.3390/pathogens12060819

47. Miller A.H., Maletic V., Raison C.L. Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression. Biol Psychiatry. 2009;65(9):732-741. doi: 10.1016/j.biopsych.2008.11.029

48. Mishchenko V.A., Goryukhina O.A., Ilyuk R.D. Changes in the bloodbrain barrier during experimental cirrhosis of the liver. Bull Exp Biol Med. 1993;116:1547-1550. doi: 10.1007/BF00785497

49. Mordvinov V.A., Furman D.P. The digenea parasite Opisthorchis felineus: a target for the discovery and development of novel drugs. Infect Disord Drug Targets. 2010;10(5):385-401. doi: 10.2174/187152610793180858

50. Murano T., Koshimizu H., Hagihara H., Miyakawa T. Transcriptomic immaturity of the hippocampus and prefrontal cortex in patients with alcoholism. Sci Rep. 2017;7:44531. doi: 10.1038/srep44531

51. Norden D.M., Trojanowski P.J., Villanueva E., Navarro E., Godbout J.P. Sequential activation of microglia and astrocyte cytokine expression precedes increased Iba-1 or GFAP immunoreactivity following systemic immune challenge. Glia. 2016;64(2):300-316. doi: 10.1002/glia.22930

52. Nunes P.T., Vedder L.C., Deak T., Savage L.M. A pivotal role for thiamine deficiency in the expression of neuroinflammation markers in models of alcohol-related brain damage. Alcohol Clin Exp Res. 2019;43(3):425-438. doi: 10.1111/acer.13946

53. Pakharukova M.Y., Zaparina O.G., Kovner A.V., Mordvinov V.A. Inhibition of Opisthorchis felineus glutathione-dependent prostaglandin synthase by resveratrol correlates with attenuation of cholangiocyte neoplasia in a hamster model of opisthorchiasis. Int J Parasitol. 2019;49(12):963-973. doi: 10.1016/j.ijpara.2019.07.002

54. Pakharukova M.Y., Lishai E.A., Zaparina O., Baginskaya N.V., Hong S.J., Sripa B., Mordvinov V.A. Opisthorchis viverrini, Clonorchis sinensis and Opisthorchis felineus liver flukes affect mammalian host microbiome in a species-specific manner. Cell Mol Life Sci. 2023;17(2):e0011111. doi: 10.1371/journal.pntd.0011111

55. Paylor R., Crawley J.N. Inbred strain differences in prepulse inhibition of the mouse startle response. Psychopharmacology. 1997;132:169-180. doi: 10.1007/s002130050333

56. Petney T.N., Andrews R.H., Saijuntha W., Wenz-Mücke A., Sithithaworn P. The zoonotic, fish-borne liver flukes Clonorchis sinensis, Opisthorchis felineus and Opisthorchis viverrini. Int J Parasitol. 2013;43(12-13):1031-1046. doi: 10.1016/j.ijpara.2013.07.007

57. Porsolt R.D., Bertin A., Jalfre M. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther. 1977;229:327-336

58. Rama Rao K.V., Jayakumar A.R., Norenberg M.D. Brain edema in acute liver failure: mechanisms and concepts. Metab Brain Dis. 2014;29(4):927-936. doi: 10.1007/s11011-014-9502-y

59. Ramos A., Mormède P. Stress and emotionality: a multidimensional and genetic approach. Neurosci Biobeh Rev. 1998;22(1):33-57. doi: 10.1016/s0149-7634(97)00001-8

60. Saijuntha W., Sithithaworn P., Petney T.N., Andrews R.H. Foodborne zoonotic parasites of the family Opisthorchiidae. Res Vet Sci. 2021; 135:404-411. doi: 10.1016/j.rvsc.2020.10.024

61. Saltykova I.V., Petrov V.A., Logacheva M.D., Ivanova P.G., Merzlikin N.V., Sazonov A.E., Ogorodova L.M., Brindley P.J. Biliary microbiota, gallstone disease and infection with Opisthorchis felineus. PLoS Negl Trop Dis. 2016;10(7):e0004809. doi: 10.1371/journal.pntd.0004809

62. Seibenhener M.L., Wooten M.C. Use of the open field maze to measure locomotor and anxiety-like behavior in mice. J Vis Exp. 2015; (96):e52434. doi: 10.3791/52434

63. Seki E., Schwabe R.F. Hepatic inflammation and fibrosis: functional links and key pathways. Hepatology. 2015;61(3):1066-1079. doi: 10.1002/hep.27332

64. Silvin A., Ginhoux F. Microglia heterogeneity along a spatio-temporal axis: more questions than answers. Glia. 2018;66(10):2045-2057. doi: 10.1002/glia.23458

65. Simon E., Obst J., Gomez-Nicola D. The evolving dialogue of microglia and neurons in Alzheimer’s disease: microglia as necessary transducers of pathology. Neuroscience. 2019;405:24-34. doi: 10.1016/j.neuroscience.2018.01.059

66. Sripa B., Thinkhamrop B., Mairiang E., Laha T., Kaewkes S., Sithithaworn P., Periago M.V., Bhudhisawasdi V., Yonglitthipagon P., Mulvenna J., Brindley P.J., Loukas A., Bethony J.M. Elevated plasma IL-6 associates with increased risk of advanced fibrosis and cholangiocarcinoma in individuals infected by Opisthorchis viverrini. PLoS Negl Trop Dis. 2012;6(5):e1654. doi: 10.1371/journal.pntd.0001654

67. Stephens A.S., Stephens S.R., Morrison N.A. Internal control genes for quantitative RT-PCR expression analysis in mouse osteoblasts, osteoclasts and macrophages. BMC Res Notes. 2011;4:410. doi: 10.1186/1756-0500-4-410

68. Stickel F., Datz C., Hampe J., Bataller R. Pathophysiology and management of alcoholic liver disease: update 2016. Gut Liver. 2017;11(2): 173-188. doi: 10.5009/gnl16477

69. Tan Y.L., Yuan Y., Tian L. Microglial regional heterogeneity and its role in the brain. Mol Psychiatry. 2020;25:351-367. doi: 10.1038/s41380-019-0609-8

70. Upadhyay R.K. Stem cell therapeutics of acute liver diseases, transplantation, and regeneration. J Cell Sci Ther. 2017;8(4):1000275. doi: 10.4172/2157-7013

71. Wang T., Chou D.Y., Ding J.Y., Fredrickson V., Peng C., Schafer S., Guthikonda M., Kreipke C., Rafols J.A., Ding Y. Reduction of brain edema and expression of aquaporins with acute ethanol treatment after traumatic brain injury. J Neurosurg. 2013;118(2):390-396. doi: 10.3171/2012.8.JNS12736

72. Yang J.Y., Xue X., Tian H., Wang X.X., Dong Y.X., Wang F., Zhao Y.N., Yao X.C., Cui W., Wu C.F. Role of microglia in ethanol-induced neurodegenerative disease: pathological and behavioral dysfunction at different developmental stages. Pharmacol Ther. 2014;144(3): 321-337. doi: 10.1016/j.pharmthera.2014.07.002

73. Yao H., Zhang D., Yu H., Yuan H., Shen H., Lan X., Liu H., Chen X., Meng F., Wu X., Zhang G., Wang X. Gut microbiota regulates chronic ethanol exposure-induced depressive-like behavior through hippocampal NLRP3-mediated neuroinflammation. Mol Psychiatry. 2023;28(2):919-930. doi: 10.1038/s41380-022-01841-y