Modulation of apoptotic signaling pathways in brain cells of adult male rats after chronic dexamethasone treatment

Deksametazon, jak sintetski glukokortikoid, se dugi niz godina koristi kao lek u tretmanu različitih bolesti poput psorijaze, adrenalne insuficijencije, bakterijalnog meningitisa, moždanih trauma, šloga, alergija, spazma bronhija, reumatidnog artritisa, itd; ali i kao pomoćni lek u hemo- i radioterapiji. Brojne studije ukazuju da deksametazon može regulisati sinaptičku plastičnost, povećati vijabilnost ćelija i njihovu proliferaciju u in vivo i in vitro uslovima. Međutim, uprkos širokoj primeni u terapijske svrhe primećeno je da deksametazon ispoljava i niz negativnih efekata u mozgu, na primer apoptozu u granularnom sloju dentatnog girusa hipokampusa kod mladih i starih pacova, kao i smanjenje kognitivnih funkcija i motornog razvoja. Usled neusklađenosti rezultata mnogih studija, proučavanje sistemskih efekata, kao i efekata deksametazona u mozgu predstavljaju interesantno polje istraživanja prvenstveno jer se efekti niskih doza ovog sintetskog glukokortikoida ne mogu ispitivati centralno usled blokiranja njegovog ulaska dejstvom MDR p-glikoproteina i/ili nekim drugim mehanizmom. Stoga, za potrebe eksperimenta, odrasli mužjaci pacova Wistar soja su podeljeni u dve grupe– kontrolne jedinke i životinje tretirane deksametazonom (100 g/kg/dan) tokom 7 uzastopnih dana. 28 h nakon završetka hroničnog tretmana životinje su žrtvovane. Sistemski efekat deksametazona je praćen promenom biometrijskih parametara (telesna masa, masa timusa i nadbubrežnih žlezdi), kao i koncentracije kortikosterona u serumu i moždanom tkivu. Kako bi se utvrdilo da li male doze deksametazona dovode do apoptoze u hipofizi, hipotalamusu, hipokampusu i prečeonoj kori korišćen je DNK fragmentacioni esej, dok se metodama histološkog bojenja hipokampusa i prečeone kore (fluoro-jade B i krezil ljubičastim) istraživao obim neuralne smrti i morfološke promene. Western blot i RT-PCR analizama su ispitivane promene u ekspresiji proteina i iRNK markera procesa smrti odnosno preživljavanja ćelija. Rezultati ove studije su pokazali da hroničan tretman malim dozama deksametazona uzrokuje hipoaktivnost hipotalamo-hipofizno-adrenalne ose, koja se ogleda u smanjenju telesne mase, mase timusa i nadbubrežnih žlezdi, kao i kortikosterona u serumu...For many years, dexamethasone, a potent synthetic glucocorticoid, has been used as a medication in the treatment of psoriasis, adrenal insufficiency, bacterial meningitis, brain trauma, stroke, allergies, bronchial spasm, rheumatoid arthritis, etc., and as a co-medicament in chemo- and radiotherapy. Numerous studies suggest that dexamethasone is able to regulate synaptic plasticity, enhance cell viability and proliferation in vivo and in vitro. However, dexamethasone exerts a number of adverse reactions in the brain, such as apoptosis in the hippocampal granular layer of dentate gyrus in young and old rats, as well as reduced cognitive and motor development. The dexamethasone-induced systemic effects and dexamethasone-provoked effects in the brain are an interesting field of research, mainly because the effects of low-dose dexamethasone treatment could not be tested directly in brain tissue due to the central blocking action of MDR p-glycoprotein and/or some other mechanism. Therefore, for the purposes of the experiment, adult male Wistar rats were divided into two groups – controls and animals treated with dexamethasone (100 g/kg/day) per 7 days. 28 h upon chronic treatment, rats from both groups were sacrificed. Late systemic effects of dexamethasone were monitored by alterations of biometric parameters (body weight, thymus and adrenal glands mass) and level of corticosterone in serum and brain tissue. Further, using DNA fragmentation assay, present study aimed to determine whether low dose dexamethasone treatment is able to cause apoptosis in the pituitary gland, hypothalamus, hippocampus and prefrontal cortex, while histological staining methods (fluoro-jade B and cresyl violet staining) were applied to investigated the extent of neuronal death and morphological changes in hippocampus and prefrontal cortex. Changes in protein and mRNA expression of cell death and cell survival markers were examined by Western blot and RT-PCR analyzes. The results obtained in this thesis revealed that chronic low dose dexamethasone treatment caused hypoactivity of hypothalamus-pituitary-adrenal axis, reflected in the reduction of body weight, thymus and adrenal glands masses, as well as levels of corticosterone in serum...