Title In vitro regeneration of primary cell cultures of cortex from postnatal grey short-tailed opossum Monodelphis domestica Title (croatian) In vitro regeneracija primarnih staničnih kultura korteksa postnatalnih oposuma Monodelphis domestica Author Marta Pongrac Mentor Jelena Ban (mentor) Committee member Ivana Munitić (predsjednik povjerenstva) Committee member Dinko Mitrečić (član povjerenstva) Committee member Miranda Mladinić Pejatović (član povjerenstva) Granter University of Rijeka Defense date and country 2021-07-23, Croatia Scientific / art field, INTERDISCIPLINARY AREAS OF KNOWLEDGE Abstract Mammalian central nervous system (CNS) begins to develop in early embryonal period. CNS has then the ability to regenerate itself, while soon after birth, regeneration stops. Opossum Monodelphis domestica is a unique model for investigating CNS regeneration because it is born at a very immature stage of CNS development. Opossum has a unique possibility to successfully regenerate spinal cord after injury in the first two weeks of their postnatal development. Regenerative capacity abruptly declines after that period and is completely lost in mature organism. Two significantly different ages of opossum at postnatal days (P)3-5 and P16-18 have different cell environment and gene profile which determine the capability of spinal cord regeneration after injury. Apart from spinal cord, opossum cortex and capability of its regeneration remain unexplored. Aim of this thesis is to establish and characterize primary cell cultures of opossum cortex, in order to explore the CNS cell composition, maturation and regeneration at a molecular basis in vitro. Immunofluorescence was used to characterize the cell composition and expression of markers linked to CNS maturation and regeneration. Scratch assay was used to study the response of cells and of their environment to injury. Calcium imaging method was used to study the maturation of cells and cell networks formed in cultures, as well as activity recovery after injury. Primary cortical cell cultures of neonatal opossum can be maintained up to a month in vitro. P3-5 cultures consist of 93% of neurons and almost 50% of cells positive for progenitor marker SOX2, while P16-18 cultures consist of 80% of neurons and 30% of SOX2+ cells. An interesting discovery is the co-expression of neuronal marker with SOX2, which was not investigated in opossum before. In vitro cortical cell culture temporal maturation correlates with the maturation of opossum cortex in vivo. Spontaneous activity of neurons and neuronal networks is observed in 7th day in vitro (DIV7) of both age groups (P3-5 and P16-18). After the injury, neurons regenerate axons which after 48 hours connect the opposite sides of the injury. A reduction in injury width, as well as glial-scar-like formation is visible in DIV10 cultures of both ages. By calcium activity, it has been shown that 48 hours after the injury, neuronal networks of the opposite sides of the injury are reconstituted and synchronized. These results contribute to understanding of opossum cortex maturation, as well as cell microenvironment on the cortex regeneration upon injury. Integration of results may contribute to better understanding of cell adaptation on pathophysiological conditions of mammalian cortex.
Abstract (croatian) Središnji živčani sustav (SŽS) sisavaca počinje s razvojem u ranom embrionalnom periodu. Tada SŽS posjeduje sposobnost regeneracije, dok se ubrzo nakon rođenja sposobnost regeneracije gubi. Oposum Monodelphis domestica jedinstven je model za istraživanje regeneracije SŽS-a jer se rađa u vrlo nezrelom stadiju razvoja SŽS-a. Oposum posjeduje sposobnost regeneracije leđne moždine nakon ozljede tijekom prva dva tjedna postnatalnog razvoja. Sposobnost regeneracije naglo pada nakon tog perioda te se u potpunosti gubi u zrelom organizmu. Dvije značajno različite dobi oposuma, postnatalni dan (P)3-5 i P16-18 sadrže različiti stanični okoliš i genski profil koji uvjetuju sposobnost regeneracije leđne moždine nakon ozljede. Za razliku od leđne moždine, korteks mozga i sposobnost njegove regeneracije u oposumu ostaju neistraženi. Cilj ovog rada je uspostava i karakterizacija primarnih staničnih kultura korteksa oposuma, kako bi se omogućilo istraživanje staničnog sastava, maturacije i regeneracije SŽS-a na molekularnoj bazi in vitro. Metodom imunofluorescencije, karakterizirao se stanični sastav i ekspresija markera povezanih s razvojem SŽS-a i regeneracijom. Izazivanjem ozlijede in vitro, istražio se odgovor stanica i staničnog okoliša na ozljedu. Funkcionalnim ispitivanjem oslikavanja kalcija, proučila se zrelost stanica i staničnih mreža formiranih u staničnim kulturama, kao i ponovno uspostavljene aktivnosti nakon ozlijede. Primarne stanične kulture korteksa neonatalnog oposuma mogu se održavati do mjesec dana u in vitro uvjetima. Stanične kulture P3-5 oposuma sadrže 93% neurona i gotovo 50% stanica pozitivnih na marker progenitora (SOX2), dok stanične kulture P16-18 oposuma sadrže 80% neurona i 30% SOX2+ stanica. Zanimljivo otkriće predstavlja koekspresija markera neurona i progenitora (SOX2), koja nije do sada istražena u oposumima. In vitro maturacija staničnih kultura korteksa vremenski korelira sa maturacijom korteksa oposuma in vivo. Spontana aktivnost neurona i mreža neurona uočena je već u 7. danu u kulturi (DIV7) za obje dobi (P3-5 i P16-18). Nakon ozljede, neuroni regeneriraju aksone koji već 48 sati nakon ozljede povezuju suprotne strane ozljede. Smanjenje promjera ozljede, kao i formiranje staničnih struktura sličnim glijalnom ožiljku je uočeno već u DIV10 kulturama obiju dobi. Oslikavanjem aktivnosti kalcija, potvrđeno je da su 48 sati nakon ozljede neuronalne mreže između suprotnih strana ozljede ponovno uspostavljene i sinkronizirane. Ovi rezultati doprinose razumijevanju maturacije korteksa oposuma, kao i utjecaju staničnog mikorookoliša na regeneraciju korteksa nakon ozljede. Integracija dobivenih rezultata mogla bi doprinijeti boljem razumijevanju prilagodbe stanica na patofiziološka stanja u korteksu sisavaca.
Keywords
primary cell cultures
opossum
injury
CNS regeneration
calcium imaging
Keywords (croatian)
primarne stanične kulture
oposum
ozljeda
regeneracija SŽS-a
oslikavanje aktivnosti kalcija
Language english URN:NBN urn:nbn:hr:193:913775 Project Number: IP-2016-06-7060 Study programme Title: Biotechnology in medicine Type of resource Text File origin Born digital Access conditions Open access Terms of use Repository Repository of the University of Rijeka, Department of Biotechnology Created on 2021-07-26 09:02:05
