Utjecaj upale na odumiranje stanica procesom apoptoze nakon ishemijske lezije mišjeg mozga

doctoral thesis

• Title (english): The effect of inflammation on apoptotic cell death after ischemic lesion of the mouse brain
• Author: Paula Josić
• Mentor: Srećko Gajović (mentor)
• Committee member: Marija Renić (predsjednik povjerenstva)
• Committee member: Leo Pažanin (član povjerenstva)
• Committee member: Lovorka Grgurević (član povjerenstva)
• Committee member: Ksenija Fumić (član povjerenstva)
• Committee member: Dinko Mitrečić (član povjerenstva)
• Granter: University of Zagreb; School of Medicine; Zagreb
• Defense date and country: 2023-04-26, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Basic Medical Sciences; Cytology, Histology and Embryology
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Basic Medical Sciences; Neuroscience
• Universal decimal classification (UDC): 616 - Pathology. Clinical medicine

Abstract

Postishemijska upala značajan je faktor u razvoju ozljede nakon moždanog udara, a TLR2 receptor jedan je od njezinih glavnih medijatora. Glavni cilj ovog doktorata bio je odrediti utjecaj upale posredovane TLR2 receptorom na apoptozu u ishemijskom okolišu.
Utjecaj TLR2 receptora istražen je na modelu ishemijskog oštećenja mišjeg mozga u životinja s normalnim (CAG-luc) i onih s onemogućenim Tlr2 genom (CAG-luc-Tlr2-/-). Oba soja ubikvitarno su izražavala luciferazni transgen. Razmjer apoptoze pratio se bioluminiscencijom zatočenim Z-DEVD-aminoluciferinom koji se cijepa i postaje bioluminiscentno aktivan nakon interakcije s pocijepanom kaspazom-3. Ishemijska ozljeda bila je uzrokovana okluzijom srednje moždane arterije, a njena progresija praćen je magnetskom rezonancom. Stanična smrt je potvrđena metodama imunofluorescencije pocijepanom kaspazom-3, protočne citometrije aneksinom-V i TUNEL esejom. Funkcionalno oštećenje izmjereno je testom neurološkog deficita.
Zatočeni Z-DEVD-aminoluciferin validiran je i procijenjen kao adekvatan supstrat za praćenje apoptoze. Longitudinalno in vivo mjerenje apoptoze izazovno je te zahtijeva kvalitetnu validaciju i normalizaciju. Nije bilo ukupne razlike u apoptozi između sojeva, ali u nedostatku TLR2 receptora postojao je izraženiji mehanizam nekroze. U akutnom periodu, CAG-luc- Tlr2-/- miševi bolje su preživljavali od CAG-luc miševa, dok u kroničnom periodu nije bilo razlike u preživljenju. Ipak, magnetna rezonanca je pokazala kako su CAG-luc-Tlr2-/- miševi su u kroničnom periodu izgubili više tkiva ipsilateralne hemisfere od CAG-luc miševa. Stoga, iako smanjena upala u akutnom periodu može biti protektivna, dugoročno narušava oporavak.

Abstract (english)

Postischemic inflammation is a significant contributor to ischemic injury development. The TLR2 receptor is one of its main mediators. The main aim of this thesis was to determine the effect of TLR2-mediated inflammation on apoptotic cell death in the ischemic environment.
The effect of TLR2 was investigated on the ischemic injury model in animals with normal Tlr2 (CAG-luc) and those with knock-out Tlr2 gene (CAG-luc-Tlr2-/-). Both strains expressed the firefly luciferase transgene ubiquitously. The scope of apoptosis was determined by the utilization of bioluminescence imaging with caged Z-DEVD-aminoluciferin, which becomes available for the bioluminescence reaction after cleavage with activated caspase-3. Middle cerebral artery occlusion was performed to produce the ischemic injury. Its progression was followed with magnetic resonance imaging. Cell death was further confirmed using immunofluorescence with activated caspase-3, flow cytometry with annexin-V and the TUNEL assay. Functional outcomes were assessed using a neurological deficit test.
Caged Z-DEVD-aminoluciferin was validated and assessed as an adequate tool for monitoring apoptosis. Longitudinal in vivo measurement of apoptosis is challenging and requires thorough validation and normalization. No total difference in apoptosis between the used strains was found. However, in the absence of the TLR2 receptor, a more pronounced mechanism of necrosis arose. In the acute period, CAG-luc-Tlr2-/- mice had better survival than CAG-luc mice. In the chronic period, there was no difference in survival between the strains. However, magnetic resonance imaging showed that CAG-luc-Tlr2-/- lost more ipsilateral hemisphere tissue than CAG-luc mice during the chronic period. Therefore, although lower levels of inflammation may be protective in the acute period post-stroke, long-term recovery is impaired.

• Keywords: TLR 2 receptor; Aneksin A5; Medijatori upale; Apoptoza; Upala; Smrt stanica; Transgeni
• Keywords (english): TLR 2 receptor; Annexin A5; Inflammation Mediators; Apoptosis; Inflammation; Cell Death; Transgenes
• Language: croatian
• URN:NBN: urn:nbn:hr:105:321199
• Project: Number: IP-2016-06-1892; Title: Multimodalni prikaz molekularnih zbivanja tijekom oporavka mišjeg mozga nakon ishemijskog oštećenja; Acronym: RepairStroke; Leader: Srećko Gajović; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: IP
• Study programme: Title: Neuroscience; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
• Type of resource: Text
• Extent: 151 str.
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-04-24
• Created on: 2023-05-26 11:37:52