Title IZOLACIJA I KARAKTERIZACIJA
IZVANSTANIČNIH VEZIKULA
CEREBROSPINALNE TEKUĆINE
BOLESNIKA S
TEŠKOM OZLJEDOM MOZGA
Title (english) ISOLATION AND CHARACTERISATION
OF EXTRACELLULAR VESICLES IN
CEREBROSPINAL FLUID DERIVED
FROM PATIENTS WITH SEVERE
TRAUMATIC BRAIN INJURY
Author Janja Kuharić
Mentor Vlatka Sotošek Tokmadžić (mentor)
Mentor Kristina Grabušić (komentor)
Committee member Alen Protić (predsjednik povjerenstva)
Committee member Željka Petelin Gadže (član povjerenstva)
Committee member Tihana Lenac Roviš (član povjerenstva)
Granter University of Rijeka Faculty of Medicine (Department of Anaesthesiology,reanimatology and Intensive Care) Rijeka
Defense date and country 2018-07-20, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Clinical Medical Sciences Anesthesiology and Reanimatology
Universal decimal classification (UDC ) 61 - Medical sciences
Thesaurus (MESH - Medical Subject Headings )
Brain Injuries
Cerebrospinal Fluid
Exosomes
Extracellular Vesicles
Abstract Cilj istraživanja: Teška ozljeda mozga je životno ugrožavajuće oštećenje strukture i
funkcije mozga. Nastaje uslijed djelovanja vanjske sile te dovodi do poremećaja svijesti
i razvoja kome. Početna ozljeda moždanog tkiva uzrokovana traumom dovodi do
pokretanja niza kaskadnih reakcija koje rezultiraju oštećenjem neurona te smanjenjem
broja sinapsi na mjestu početne ozljede, ali i udaljenim dijelovima mozga. Osnova
oporavka se zasniva na stvaranju novih i restrukturiranju postojećih sinapsi što
podrazumijeva složenu i visoko reguliranu komunikaciju između neurona i glija stanica.
Usprkos brojnim istraživanjima, o navedenim mehanizmima ne zna se mnogo. S
obzirom da su izvanstanične vezikule bitan posrednik međustanične komunikacije, cilj
istraživanja bio je izolirati i karakterizirati izvanstanične vezikule cerebrospinalne
tekućine (CSF) bolesnika s teškom ozljedom mozga tijekom prvih sedam dana.
Ispitanici i metode: Izolacija izvanstaničnih vezikula CSF-a bolesnika s teškom
ozljedom mozga provedena je ultracentrifugiranjem CSF-a u slijedu, a karakteristike i
vremenske promjene izvanstaničnih vezikula su ispitane metodama elektronske
mikroskopije, analizom nanočestica u otopini, western blot te enzimskoimunokemijskom
analizom.
Rezultati: Prisutnost izvanstaničnih vezikula u CSF-u vizualizirana je elektronskom
mikroskopijom i potvrđena biomarkerima izvanstaničnih vezikula, Flotilinom-1 i 2, ADP
ribozilacijskim čimbenicima 1 i 6 (Arf1 i Arf6) te Ras-povezanim bjelančevinama Rab5b
i Rab7a (Rab5b i Rab7a). Analiza nanonočestica u CSF-u je pokazala najvišu
koncentraciju izvanstaničnih vezikula u vremenskoj točki d1 te značajni porast u
njihovoj veličini u vremenskoj točki d4-7. Koncentracije biomarkera izvanstaničnih
vezikula i bjelančevina vezanih uz sinaptičku plastičnost mjerenih enzimatskoimunokemijskom
analizom CSF-a pokazale su da je Flotilin-1 prisutan u trećine
bolesnika u zadnjoj vremenskoj točki uzorkovanja, a da je loš ishod liječenja povezan
s padom u koncentraciji Arf6 i odgođenim porastom u koncentraciji Rab7a.
Zaključak: Teška ozljeda mozga dovodi do značajnih promjena u veličini, koncentraciji
i bjelančevinskom sastavu izvanstaničnih vezikula CSF-a.
Abstract (english) Objectives: Severe traumatic brain injury (TBI) is a life threatening intracranial injury
caused by external force that leads to altered consciousness and coma. It has complex
pathophysiology which involves initial injury occurring at the moment of trauma,
followed by series of events that lead to further neuronal damage and reduction in the
number of synapses at places distant from the initial injury site. Based on the current
knowledge, the foundation of recovery is plasticity response capable of restoring lost
synaptic contacts. This requires complex and highly regulated communication between
neurons and glial cells. Despite extensive studies, underlying mechanisms are widely
unknown. Extracellular vesicles (EV) are newly recognized part of intercellular
communication with possible involvement in pathogenesis of TBI. The aim of the study
was to isolate and characterize EV in ventricular cerebrospinal fluid (CSF) of patients
with severe TBI during the first seven days after the injury.
Patients and methods: Isolation of EVs was conducted using sequential
ultracentrifugation and characterization was performed using electron microscopy,
nanoparticle tracking analysis, western blot and enzyme-immunochemical analysis.
Results: EVs in TBI-CSF were visualized by electron microscopy and confirmed by
immunoblotting for Flotilin-1 and 2, ADP ribosylation factors 1 and 6 (Arf1 and Arf6)
and Ras-related protein Rab5b and Rab7a (Rab5b and Rab7a). Using nanoparticle
tracking analysis, the highest range in EV concentration was detected at day 1 after
injury and significant increase in EV size was seen at days 4-7. CSF concentrations of
EV biomarkers and neuroregeneration associated proteins Flotillin-1, Arf6 and Rab7a
were monitored by enzyme-linked immunosorbent assays. Flotillin-1 was detected
solely in TBI-CSF in three of the tested patients at final sampling points. Unfavorable
outcomes included decreasing Arf6 concentrations and a delayed Rab7a
concentration increase in CSF.
Conclusion: TBI induces significant alterations in EV size, concentration, and protein
composition during the first 7 days after the injury. These early variations in EV
properties offer insight into ongoing neuronal processes after a severe TBI with Rab7a,
Arf6, and Flotillin-1 having the potential to indicate functional outcome of patients
Keywords
Cerebrospinalna tekućina
Egzosomi
Izvanstanične vezikule
Mikrovezikule
Teška ozljeda mozga
Keywords (english)
Cerebrospinal fluid
Exosomes
Extracellular vesicles
Microvesicles
Traumatic brain injury
Language croatian
URN:NBN urn:nbn:hr:184:025473
Study programme Title: Biomedicine Postgraduate (doctoral) study programme Study programme type: university Study level: postgraduate Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
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Created on 2019-01-28 10:37:54