Utišavanje ATF3 u primarnim kulturama korteksa neonatalnog oposuma Monodelphis domestica

Abstract

The mammalian central nervous system (CNS) develops primarily during the embryonal period, but final structuring and tuning occurs postnatally. Before or soon after birth, the mammalian CNS loses its ability to fully regenerate after injury. Therefore, neuroregeneration studies are mostly conducted on rodent embryos (mice or rats) or derived in vitro preparations, such as primary cell or tissue cultures. However, an intrauterine surgical procedure is necessary to access rodent embryos, which usually means sacrificing the mother - an ethically questionable issue. Therefore, to study regenerative mechanisms of the CNS, we have used an alternative experimental animal: the grey South American short-tailed opossum (Monodelphis domestica), a marsupial without a pouch. Opossum neonates are very immature, and their CNS develops mostly postnatally, with the cortical plate not observed until postnatal day (P)3-5. Therefore, harming the mother is not necessary in order to investigate CNS development in opossums. Furthermore, opossums are unique among mammals in their ability to successfully regenerate the spinal cord after injury during the first two weeks of their life. After that period, the regenerative capability of their spinal tissue is lost. Thus, the first aim of this work was to characterize primary cortical cell cultures obtained from opossums at two developmentally different ages, P3-5 and P16-18, using immunofluorescent microscopy. The results showed a high percentage of neurons in primary cultures derived from both P3-5 and P16-18 opossums (>96% and >80% at DIV7, respectively). The second aim was to genetically silence activating transcription factor 3 (ATF3) in opossum primary cortical cultures, via transfection with specific siRNA molecules, to further investigate its role in neuroregeneration. The cell transfection protocol was optimized to reach the optimal transfection efficiency, and the ATF3 gene and protein levels were assessed with quantitative real-time PCR and Western blot, respectively. Altogether, the results indicate that primary cortical cultures obtained from opossum M. domestica are favorable preparation that can be used to investigate mechanisms underlying CNS regeneration, particularly the involvement of regeneration-related transcription factors, such as ATF3.Središnji živčani sustav (SŽS) sisavaca se razvija primarno tijekom embrionalnog razvoja, a funkcionalno sazrijevanje živčanih mreža i procesa događa se postnatalno. Prije ili nedugo nakon rođenja, sisavci gube sposobnost potpune regeneracije SŽS-a nakon ozljede. Većina istraživanja regeneracije SŽS-a provodi se na embrijima glodavaca (miševa ili štakora) ili in vitro preparatima, poput primarnih staničnih ili tkivnih kultura. Embrijima se ne može pristupiti bez intrauterine operacije, a to obično podrazumijeva žrtvovanje majke što je etički upitno. Iz tog razloga, za istraživanje regenerativnih mehanizama SŽS-a odlučili smo se za alternativni organizam: sivi kratkorepi oposum (Monodelphis domestica), tobolčar bez posteljice. Mladunci oposuma se rađaju vrlo nezreli, te se njihov SŽS većinom razvija postnatalno, kortikalnu ploču, primjerice, nije moguće uočiti do postnatalnog dana (P)3-5. Dakle, razvoj SŽS-a se kod oposuma može istraživati bez potrebe da se pritom šteti majci. Nadalje, oposumi su posebni među sisavcima jer u prva dva tjedna života mogu uspješno regenerirati leđnu moždinu nakon ozljede. Nakon tog razdoblja, mogućnost regeneracije moždanog tkiva se gubi. Stoga, prvi cilj ovog rada je bio pomoću imunofluorescentne mikroskopije opisati primarne stanične kulture korteksa pripremljene od dvije razvojno različite dobi oposuma, starih P3-5 i P16-18. Rezultati su pokazali visok postotak neurona u staničnim kulturama pripremljenih od P3-5 (>96%) i P16-18 (>80%) oposuma nakon 7 dana u kulturi. Drugi cilj bio je genetski utišati aktivirajući transkripcijski faktor 3 (ATF3) u primarnim staničnim kulturama korteksa oposuma koristeći metodu transfekcije sa specifičnim siRNA molekulama, kako bi se pobliže istražila njegova uloga u neuroregeneraciji. Protokol je bio optimiziran kako bi se postigla optimalna učinkovitost transfekcije, a razine ATF3 mRNA i proteina, bile su provjerene kvantitativnim PCR-om u realnom vremenu, odnosno Western blot-om. Zaključno, rezultati ovog rada ukazuju na to da su primarne stanične kulture korteksa oposuma M. domestica poželjan preparat za istraživanje mehanizama regeneracije SŽS-a, osobito u istraživanju uloge transkripcijskih faktora povezanih s regeneracijom kao što je ATF3