Rano povećanje ekspresije mangan superoksid dismutaze nakon eksperimentalne povrede mozga pacova

Abstract

Povreda mozga obuhvata primarnu povredu, koja nastaje kao rezultat neposrednog mehaničkog oštećenja tkiva i sekundarnu povredu koja se javlja tokom perioda od narednih nekoliko dana. Iako još uvek nisu poznati svi detalji mehanizama koji dovode do sekundarne povrede, nekoliko metaboličkih promena, uključujući i povećanje produkcije reaktivnih kiseoničnih vrsta (ROS) dovedeno je u vezu sa patofiziologijom sekundarne povrede. Budući da su mitohondrije primarno unutarćelijsko mesto nastanka ROS, pretpostavlja se da mangan superoksid dismutaza (MnSOD, SOD2) ima presudnu zaštitnu ulogu u antioksidativnim mehanizmima odbrane i preživljavanju nervnih ćelija nakon povrede. Stoga je u ovom radu praćena ekspresija MnSOD u modelu unilateralne ablacije korteksa pacova, sa ciljem da se rasvetli njena uloga u ranim događajima nakon povrede mozga. Svi eksperimenti izvedeni su na pacovima soja Wistar starim tri meseca. Životinjama je pod anestezijom Zoletilom, uklonjen senzomotorni korteks na levoj strani, pažljivim usisavanjem tkiva kroz polipropilenski vrh. Životinje se žrtvovane 0, 4, 24 i 72 časa nakon povrede i tkivo levog (LCtx) i desnog (RCtx) korteksa disecirano je i iskorišćeno za pripremu tkivnih homogenata. Za svaku eksprimentalnu tačku, korišćena je grupa lažno operisanih životinja, dok su neoperisane, intaktne životinje primenjene kao fiziološka kontrola. Intenzitet signala ekspresije iRNK i proteina kod povređenih životinja izražen je u odnosu na lažno-operisane životinje, dok je intenzitet dobijen kod intaktnih životinja arbitrarno definisan kao 1.00. RtPCR analiza demonstrirala je da u LCtx dolazi do rane indukcije iRNK za MnSOD između 4 i 24 h nakon povrede. Uočeno vremenski-zavisno povećanje iRNK za MnSOD bilo je najveće 4 sata nakon povrede. Saglasno tome, imunoblot analiza pokazala je da je ekspresija MnSOD proteina u LCtx značajno povećana tokom prva četiri sata nakon povrede. Ekspresija iRNK i MnSOD proteina vraća se na fiziološki nivo 72 sata nakon povrede. S druge strane, ekspresija MnSOD, kako na nivou iRNK, tako i na nivou proteina, ne menja se u RCtx tokom celog eksperimentalnog perioda. Rezultati ovog rada ukazuju da povreda mozga dovodi do brzog i značajnog povećanja ekspresije MnSOD na mestu povrede, najverovatnije kao deo odgovora na oksidativni stres uzrokovan primarnom povredom. Budući da MnSOD predstavlja prvu liniju odbrane od superoksid anjon radikala koji nastaju u mitohondrijama, ovi nalazi mogu doprineti boljem razumevanju uloge MnSOD u procesu oporavka nakon povrede.

Brain injury consists of primary injury that is the result of immediate mechanical damage and secondary injury that evolves over a period of minutes and days. The precise mechanisms underlying secondary injury are not well understood, however several metabolic dearangements, including increased generation of reactive oxygen species (ROS) have been implicated in the pathophysiology following brain damage. Since mitochondria are the major subcellular site of ROS generation, manganese superoxid dismutase (MnSOD, SOD2), a potent scavanger of superoxide radicals could have critical cytoprotective role in the antioxidant defence mechanism and neuronal survival after brain damage. Thus, in the present study we have evaluated expression of MnSOD to address its role during early events of brain injury using a model of unilateral cortical ablation in rat. Experiments were performed on three-month old Wistar male rats. The sensomotory cortex was unilaterally removed on the left side by gentle suction aspiration through polypropilene tip under the Zoletil anesthesia. Animals were sacrificed 0, 4, 24 and 72 hours after the surgery and left (LCtx) and right (RCtx) cortical tissues were immediately isolated for tissue homogenate preparations. For each time point, another group of aged-matched animals was used as sham-operated controls, whereas non-operated, intact animals were used as a physiological control. Signal intensities obtained for MnSOD mRNA and protein expression in injured animals were expressed relative to that obtained for sham-operated animals at each time point after the surgery, whereas signal intensity obtained for intact control was arbitrarily defined as 1.00. RtPCR analysis showed a rapid induction of MnSOD mRNA in LCtx between 4 and 24 h after the injury. Observed time-dependent increase in MnSOD mRNA was maximal 4 hours after the injury compared to the level induced by sham operation alone. Accordingly, immunoblot analysis demonstrated increased expression of MnSOD protein in LCtx up to 4 h after the injury. 72 hours after the injury MnSOD mRNA and protein expression return to the level of the intact control. On the other hand, MnSOD mRNA and protein expression remained unaffected in the RCtx at all time points after the surgery. In conclusion, the result of this study demonstrate that brain injury induce rapid and marked increase in MnSOD expression at the site of injury, most likely as a protective response after oxidative stress initiated by primary brain damage. Since MnSOD provides the first line of defense against superoxide generated in mitochondria, these findings may contribute to a better understanding of role MnSOD in the recovery process following brain injury.