Title Učinci preopterećenja željezom u eksperimentalnom autoimunosnom encefalomijelitisu Title (english) Effects of iron overload in experimental autoimmune encephalomyelitis Author Božena Ćurko-Cofek Mentor Vesna Barac-Latas (mentor)Committee member Gordana Župan (predsjednik povjerenstva)Committee member Sabina Rabatić (član povjerenstva)Committee member Lidija Tuškan-Mohar (član povjerenstva)Committee member Vesna Barac-Latas (član povjerenstva)Granter University of Rijeka Defense date and country 2017-11-09, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences ThesaurusMESH - Medical Subject Headings )
Encephalomyelitis, Autoimmune, Experimental
Lipid Peroxidation
Multiple Sclerosis
Iron Overload
Sex
Abstract Cilj istraživanja
Glavni cilj istraživanja bio je utvrditi i usporediti učinke preopterećenja željezom u Dark Agouti (DA) štakora oba spola u modelu kronično-relapsirajućeg eksperimentalnog autoimunosnog encefalomijelitisa (CR-EAE) te u štakora oba spola navedenog soja u kojih nije bila učinjena indukcija CR-EAE-a. Pratili smo utjecaj preopterećenja željezom na i) homeostazu željeza i funkciju jetre, ii) razinu oksidacijskog oštećenja lipida u jetri, mozgu i kralježničnoj moždini, iii) izražaj bjelančevina akutnog stresnog odgovora u tkivima jetre, mozga i kralježnične moždine. U životinja s CR-EAE-om dodatno smo istraživali iv) pojavu i intenzitet kliničkih znakova bolesti i smrtnost te v) opseg demijelinizacije u mozgu i kralježničnoj moždini. Posebna pozornost posvećena je utvrđivanju postojanja spolnog dimorfizma po svim ispitivanim parametrima.
Materijal i metode
Istraživanja su provedena na mužjacima i ženkama štakora DA soja, dobi 6-8 tjedana koji su bili preopterećeni željezom. U dijela životinja bio je induciran CR-EAE aplikacijom encefalitogena. Za analize je uzet serum te tkiva jetre, mozga i kralježnične moždine štakora. U serumu su metodom ELISA-e (engl. enzyme-linked immunosorbent assay) određene koncentracije feritina i hepcidina, a jetrene transaminaze bikromatskim spektrometrijskim mjerenjem. Spektrometrijske metode korištene su i za određivanje koncentracije željeza i malondialdehida (MDA) u tkivima. Distribucija željeza u tkivima prikazana je histokemijskim bojanjem, a promjene u izražaju bjelančevina 4-hidroksinonenala (4-HNE), hepcidina, metalotioneina I/II (MT I/II) i glikoproteina 96 (gp96) imunohistokemijskim bojanjem parafinskih rezova tkiva. Demijelinizacijske promjene u tkivima mozga i kralježnične moždine prikazane su metodom Klüver-Barrera, a metodom imunofluorescencije dodatno je proučen izražaj hepcidina i MT I/II u neuronima kralježnične moždine.
Rezultati
U DA štakora koji su bili preopterećeni željezom, a u kojih nije bio induciran CR-EAE, pronađene su spolne razlike u nekim od istraživanih parametara. Ženke su imale jači izražaj hepcidina i MT I/II u svim ispitivanim tkivima, a mužjaci jači izražaj gp96 u tkivima jetre i mozga.
Rezultati preopterećenja željezom pokazali su puno izraženije spolne razlike u štakora s CR-EAE-om. Preopterećenje željezom utjecalo je na klinički tijek bolesti na način da je u ženki došlo do ranijeg početka i ranijeg razvoja akutne faze bolesti, a u mužjaka je ubrzana pojava relapsa te značajno povećana smrtnost. Ženke s CR-EAE-om su pokazale veću sklonost pohrane željeza u obliku feritina, što je bilo potaknuto i većom koncentracijom hepcidina. Produkti lipidne peroksidacije (MDA, 4-HNE) bili su jače izraženi u jetri ženki, a u mužjaka u mozgu i kralježničnoj moždini, što su pratile jače razvijene demijelinizacijske promjene, osobito u kralježničnoj moždini. Spolnim razlikama pridonio je i jači izražaj citoprotektivnih elemenata (hepcidina, MT I/II, gp96) u tkivima ženki s CR-EAE-om preopterećenih željezom u odnosu na odgovarajuću skupinu mužjaka, osobito u neuronima mozga i kralježnične moždine.
Zaključak
Dobiveni rezultati ukazuju da preopterećenje željezom različito utječe na mehanizme koji reguliraju homeostazu željeza u štakora DA soja s i bez znakova CR-EAE-a, na periferiji i u središnjem živčanom sustavu. Preopterećenje željezom utječe i na dinamiku CR-EAE-a u mužjaka i ženki DA štakora. Ono u mužjaka rezultira većom razinom oksidacijskog oštećenja lipida, a u ženki jačim izražajem citoprotektivnih bjelančevina, što bi moglo biti u svezi s ispoljenim spolnim razlikama u početku bolesti, intenzitetu kliničkih znakova i smrtnosti
Abstract (english) Objectives
The main goal of our research was to determine and compare effects of iron overload on male and female Dark Agouti (DA) rats with chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE) and male and female rats without induction of CR-EAE. We tracked the influence of iron overload on the i) iron homeostasis and liver function, ii) level of oxidative dammage of lipids in the liver, brain and spinal cord, iii) expression of acute stress response proteins in the liver, brain and spinal cord tissues. Additionaly, in the animals with CR-EAE we determined iv) onset and intensity of clinical signs and mortality rate and v) demyelination changes in the brain and spinal cord. Particular attention has been devoted to determining the existence of sexual dimorphism in all examined parameters.
Material and Methods
The study was performed on male and female iron overloaded DA rat strain, aged 6-8 weeks. In the group of rats with CR-EAE the disease was induced with encephalitogen. To perform the analyses, we took the serum and liver, brain and spinal cord tissues. Serum concentrations of ferritin and hepcidin were determined using enzyme-linked immunosorbent assay (ELISA) and for liver transaminases bichromatic spectrometrical method was used. Spectrometrical methods were also used for iron and malondialdehyde (MDA) concentrations in the tissues. To show iron distribution in tissues we used histochemical staining and for the changes in expression of 4-hydroxynonenal (4-HNE), hepcidin, metallothioneins I/II (MT I/II) and glycoprotein 96 (gp96) immunohistochemical staining of paraffin tissue slides were used. Demyelinating changes in the brain and spinal cord tissues were shown using Klüver-Barrera method and for additional analysis of hepcidin and MT I/II expression in spinal cord neurons we used immunofluorescence method.
Results
In the iron overloaded DA rats without CR-EAE the results point to the sex differences in some of the examined parameters. Female rats had higher expression of hepcidin and MT I/II in all analysed tissues while male rats had higher expression of gp96 in the liver and brain tissues.
The results of iron overload showed much bigger sex-related differences on rats with CR-EAE. Iron overload affected the clinical course of disease in the way that female rats had earlier onset and acute phase of disease while male rats had earlier appearance of relapse and higher mortality rate. Female rats with CR-EAE showed bigger tendency to deposit the iron in the form of ferritin which was induced by the higher concentration of hepcidin. Lipid peroxidation products (MDA, 4-HNE) had a higher expression in the liver of female rats and mail rats had higher expression in the brain and spinal cord which was related to more developed demyelinating changes, especially in the spinal cord. Stronger expression of cytoprotective elements (hepcidin, MT I/II, gp96) in the tissues of iron overloaded female rats with CR-EAE, especially in the neurons of the brain and spinal cord, also contributed to the sex-related differences.
Conclusion
The results imply that iron overload has a different impact on mechanisms that regulate iron homeostasis in the DA rat strain with or without CR-EAE, on the periphery and in the central nervous system. Iron overload also affects the dynamic of CR-EAE in male and female DA rats. It increases the level of oxidative damage of lipids in male rats while the cytoprotective mechanisms are more expressed in female rats which could contribute to the sex-related differences in the onset of disease, intensity of the clinical signs and mortality rate
Keywords
Eksperimentalni autoimunosni encefalomijelitis
Lipidna peroksidacija
Multipla skleroza
Preopterećenje željezom
Spol
Keywords (english)
Encephalomyelitis
Autoimmune
Experimental
Iron Overload
Lipid Peroxidation
Multiple Sclerosis
Sex
Language croatian URN:NBN urn:nbn:hr:184:627673 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2018-01-09 08:59:25
