In vitro study to investigate effect of hypoxia on astroglial and neuronal co-cultures

master's thesis

• Title (croatian): In vitro studija utjecaja hipoksije na uzgoj mješovite kulture astroglije i neurona
• Author: Rana Tahir Rasool
• Mentor: Dinko Mitrečić (mentor)
• Committee member: Marina Radmilović (predsjednik povjerenstva)
• Committee member: Dinko Mitrečić (član povjerenstva)
• Committee member: Davor Ježek (član povjerenstva)
• Granter: University of Zagreb; School of Medicine; (Department of Histology and Embryology); Zagreb
• Defense date and country: 2018-07-13, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Basic Medical Sciences; Cytology, Histology and Embryology

Abstract

INTRODUCTION: Stroke is the second most common cause of death in the world and 85% of strokes are ischemic due to obstruction of blood vessels. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. There is an increasing interest in developing new therapies for ischemic stroke due to short therapeutic window and lack of
currently available treatments. Astrocytes provide a major role in brain’s response to ischemic injury by providing antioxidant protection, growth factors and glutamate clearance. A detailed understanding of astrocyte-neuron interaction can provide valuable therapeutic targets. This study aims to use astroglial-neuronal co-cultures to investigate affect of hypoxia on interaction between astrocytes and neurons. ----- METHODS: Primary astrocytes were obtained from mouse newborns and NSCs from E14.5 mouse embryos. Astrocytes and NSCs were seeded together to develop astroglial-neuronal co-cultures, which were incubated in normoxic and hypoxic conditions. Co-cultures were characterized using live imagining and immunocytochemistry, quantification of cellular motility and relative gene expression of apoptotic (caspase 8) and necroptotic marker (MLKL) was performed using live cell tracing and rt-PCR respectively. ----- RESULTS: The characterization of co-cultures in hypoxic conditions showed NSCs differentiation into unipolar and bipolar neurons and presence of flat and epithelioid astrocytes. The average and maximum speed of cells in astroglial-neuronal co-cultures was higher in hypoxic conditions, but less than NSCs single culture in normoxia. Relative gene expression of caspase 8 was higher than MLKL in both hypoxic and normoxic conditions. ----- DISCUSSION: There was better differentiation and survival of NSCs in astroglial-neuronal co-cultures in hypoxic conditions. This was also reflected in better cellular motility of astroglialneuronal co-cultures in hypoxic conditions. Caspase 8 and MLKL exhibited reciprocal relationship of expression in both normoxic and hypoxic conditions. Although we observed
difference in expression of caspase 8 and MLKL in normoxic and hypoxic conditions, more samples will be needed to define precise levels of statistical significance of this finding. ----- CONCLUSION: In this work we observed responses of astrocytes and neurons in astroglialneuronal co-cultures, which were fairly consistent with what happens in ischemia. More studies will be required to characterize and understand observed phenomena in the astroglialneuronal co-cultures.

Abstract (croatian)

UVOD: Moždani udar je drugi najčešći uzrok smrti u svijetu. U 85% slučajeva se radi o ishemijskom moždanom udaru uzrokovanom prekidom protoka krvi, a time glukoze i kisika, kroz krvnu žilu, što uzrokuje energetski disbalans i smrt neurona. Trenutno dostupni terapijski pristupi mogu se primjeniti u ograničenom vremenskom razdoblju te stoga postoji
potreba za novim pristupima u liječenju ishemijskog moždanog udara. Astrociti imaju važnu ulogu u odgovoru na ishemiju mozga jer pružaju antioksidativnu zaštitu, proizvode faktore rasta te uklanjaju glutamat. Bolje poznavanje interakcija između neurona i astrocita moglo bi iznjedriti neke nove terapijske mete. Cilj ovog rada je bio uspostaviti kokulture astrocita i neurona te istražiti utjecaj hiposksije na interakciju astrocita i neurona. ----- METODE: Primarni astrociti izolirani su iz novorođenih miševa, dok su živčane matične stanice izolirane iz embrija miševa starih 14.5 dana. Astrociti i živčane matične stanice su nasađeni zajedno kako bi se dobila mješovita kultura, uzgajana u u normalnim uvjetima te u uvjetima hipoksije. Mješovite kulture su analizirane imunohistokemijski, praćena je mobilnost stanica i analizirana je ekspresija apoptotičkog (Casp8) i nekroptotičkog markera (Mlkl) metodom qRT-PCR. ----- REZULTATI: Karaterizacija mješovitih kultura u hipoksičnim uvjetima pokazala je da su se živčane matične stanice diferencirale u unipolarne i bipolarne neurone te je pokazana prisutnost plosnatih i elipsoidnih astrocita. Vrijednosti prosječne i maksimalne brzine stanica u mješovitim kulturama su bile veće u hipoksičnim uvjetima, ali manje od onih zabilježenih u kulturi samih živčanih matičnih stanica u normalnim uvjetima. Relativna ekspresija gena Casp8 bila je veća od ekspresije Mlkl i u hipoksičnim i normalnim uvjetima. ----- DISKUSIJA: U hipoksičnim uvjetima zabilježena je bolja diferencijacija i preživljenje živčanih matičnih stanica te bolja mobilnost stanica u kokulturi. Iako je zabilježena razlika u relativnoj ekspresiji gena Casp8 i Mlkl u normalnim i hipoksičnim uvjetima, za određivanje razine statističke značajnosti će biti potrebno imati veći broj bioloških replika. ----- ZAKLJUČAK: U mješovitoj kulturi astrocita i neurona smo opazili promjene koje se inače dešavaju u ishemiji. Potrebna su nova istraživanja na ovim stanicama koja će dovesti do boljeg razumijevanja interakcija prisutnih u hipoksiji.

• Keywords: astrocytes; neuronal stem cells (NSCs); astroglial-neuronal co-cultures; hypoxia; cell motility; caspase 8; MLKL; real-time PCR
• Keywords (croatian): astrociti; NSC (živčane matične stanice); kokultura astrocita i neurona; mobilnost stanica; Casp8; Mlkl; qRT-PCR
• Language: english
• URN:NBN: urn:nbn:hr:105:812487
• Study programme: Title: Medicine (in English language); Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: doktor/doktorica medicine (doktor/doktorica medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-04-30 12:26:56