The effect of lithium on the neurotransmitter and antioxidant status in the perfrontal cortex and hippocamus of the rats exposed to restraint stress

Cilj istraživanja je bio da se detaljnije prouče molekulski mehanizmi delovanja litijuma na neurotransmiterski i antioksidativni status u prefrontalnom korteksu i hipokampusu stresiranih životinja. Specifični cilj rada bio je da se ispita uticaj ponavljanog stresa fizičkog sputavanja u trajanju od 14 dana, kao i delovanje litijuma kod pacova izlaganih 14 dana ponavljanom stresu fizičkog sputavanja na: količinu dopamina (DA) i noradrenalina (NA), ekspresiju gena za tirozin hidroksilazu (TH) i dopamin β hidroksilazu (DBH) (enzime koji učestvuju u biosintezi DA i NA), ekspresiju gena za dopaminske i noradrenalinske transportere odgovorne za preuzimanje, skladištenje i odpuštanje DA i NA (DAT, NET i VMAT 2), aktivnost i količinu enzima odgovornih za razgradnju DA i NA (MAO A, MAO B i COMT), ekspresiju gena za enzime antioksidativne zaštite (SOD 1, SOD 2, CAT, GPx i GR), ekspresiju gena za BDNF u prefrontalnom korteksu i hipokampusu, koncentraciju ACTH i CORT u plazmi i ponašanje eksperimentalnih životinja. U eksperimentima je korišćen ponavljani stres fizičkog sputavanja, izvođen na taj način što je pacov podvrgnut stanju nepokretnosti smeštanjem u plastične tube dimenzija 25x7 cm. Pacovi su podvrgnuti opisanom stresu po dva sata tokom 14 dana u različito vreme u periodu od 9-13 h. Rastvor litijum-hlorida (LiCl) je pacovima injeciran intraperitonealno jednom dnevno 14 dana. Početna doza litijuma je bila 1,5 mEq/kg i primenjena je prva dva dana. Zatim je povećana na 2,3 mEq/kg sedam dana, pa na 3 mEq/kg pet dana. Ovakav protokol davanja LiCl pacovima obezbeđuje koncentracije litijuma u plazmi koje su, u poređenju sa pacijentima sa bipolarnim poremećajem, iznad minimalne terapeutske koncentracije (i. e. 0,4 mM) tokom perioda terapije. U ovom istraživanju korišćene su sledeće metode: 1. Metoda za kvantifikaciju ekspresije TH, DBH, DAT, NET, VMAT 2, SOD 1, SOD 2, CAT, GPx, GR i BDNF gena (RТ-PCR-а u realnom vremenu sa TaqMan probama); 2. Metoda za kvantifikaciju sinteze TH, DBH, DAT, NET, VMAT 2, COMT, SOD 1, SOD 2, CAT, GPx, GR i BDNF proteina (Wеstern blot); 3. Metode za određivanje aktivnosti enzima MAO A i MAO B (Amplex Red Monoamine Oxidase Assay kit; molecular Probes), SOD 1, SOD 2, CAT, GPx i GR (Randox Laboratories, Crumlin, UK; metoda po Claiborn-u; metoda po Maral-u; metoda po Glatzle-u); 4. Metoda za određivanje DA i NA u tkivu (kit 3-CAT Research ELISA kits BA E-5600, Labor Diagnostica Nord, Nordhorn, Germany); 5. ELISA еsеј, metoda za određivanje CORT u plazmi (OCTEIA EIA kit, Immunodiagnostic Systems Inc.); 6 Hemiluminiscentna metoda za određivanje ACTH u plazmi pomoću automatskog analizatora...The aim of this research was to investigate the molecular mechanisms underlying the effects of lithium on neurotransmitter and antioxidant status in prefrontal cortex and hippocampus of the stressed animals. The specific aim of the work was to investigate the effect of chronic restraint stress in a period of 14 days and also the effect of lithium administration on: the amounts of dopamine (DA) and noradrenaline (NA), the gene expression of tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) (biosynthetic enzymes for DA and NA), the gene expression of transport proteins for uptake, storage and release of DA and NA (DAT, NET and VMAT 2), the activity and amount of DA and NA degrading enzymes (MAO A, MAO B and COMT), the gene expression of antioxidant enzymes (SOD 1, SOD 2, CAT, GPx and GR), the gene expression of BDNF in prefrontal cortex and hippocampus, the plasma concentrations of ACTH and CORT, as well as on the behavior of experimental animals. In our experiments the rats were subjected to chronic restraint stress by being placed in the plastic tubes sized 25x7 cm, two hours a day for 14 days, at different times in the period from 9 to 13 h. Lithium-chloride solution (LiCl) was injected to the animals intraperitoneally once a day during 14 days. The initial dose of lithium of 1,5m Eq/kg was applied in the first two days. The dose was increased to 2,3 mEq/kg during the next 7 days and to 3 mEq/kg during the following 5 days. This protocol of LiCl administration provided the concentrations of lithium in the plasma of rats to be higher than the minimal therapeutic concentration (i.e. 0,4 mM) in the patients with bipolar disorder. The following methods were used in this investigation: 1. The expressions of TH, DBH, DAT, NET, VMAT 2, SOD 1, SOD 2, CAT, GPx, GR and BDNF were quantified by using real time RT-PCR with TaqMan assays. 2. Western blot analysis was used to quantify the synthesis of TH, DBH, DAT, NET, VMAT 2, COMT, SOD1, SOD 2, CAT, GPx, GR and BDNF protein. 3. Methods for evaluation of MAO A and MAO B enzyme activity (Amplex Red Monoamine Oxidase Assay kit; molecular Probes), enzyme activities of SOD 1, SOD 2, CAT, GPx i GR (Randox Laboratories, Crumlin, UK; method by Claiborn-u; method by Maral-u; method by Glatzle-u); 4. Methods for measuring DA i NA in the tissue (kit 3-CAT Research ELISA kits BA E-5600, Labor Diagnostica Nord, Nordhorn, Germany); 5. ELISA assay, the method for measuring CORT in the plasma (OCTEIA EIA kit, Immunodiagnostic Systems Inc.); 6. Hemiluminiscent method for measuring of ACTH in the plasma by automatic analyzer..