Aktivnost komponenti adenozinskog signalnog sistema astrocita u modelu moždane povrede in vivo i in vitro
Activity of the astrocytes' adenosine signaling system components in model of traumatic brain injury in vivo and in vitro
Doctoral thesis (Published version)
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Reactive astrogliosis is a hallmark of traumatic brain injury (TBI), which, among the others alterations, causes changes in purinergic signaling. Due to its neuroprotective features, fluctuations of adenosine extracellular concentration are particularly important aspect of purinergic signaling in brain pathology. Hence, herein given thesis aimed to investigate expression and function of astrocytes’ adenosine signaling system components after brain injury in vitro and in vivo, with special regard to the role of equilibrative nucleoside transporters (ENT). In vivo study, performed on a model of cortical stub injury of rat forebrain, showed that injury caused dynamic changes in expression of ENTs, ectonucleotidases, and adenosine A1 receptor. Moreover, injury induced cell redistribution of ENT1/2 and upregulation of transporters on reactive astrocytes, which is especially pronounced seven day after the impact. The role of astrocytes in orchestration of adenosine signaling system after the injury was examined in more details in vitro, after scratch wound injury of astrocytic monolayer. Results have shown that scarification induced upregulation of ENT1 and ENT2 in later time points. Biphasic alteration in expression of e-5NT was shown in early downregulation followed by upregulation of the enzyme in later time points after the induction of scratch wound. Beside, scarification of astrocytic monolayer caused changes in concentration of adenosine and its metabolites in extracellular medium. The rise of adenosine concentration was noted early after the injury, which was followed by drop of the concentration in later time points examined. Blocking of ENT with dipyridamole (DPM) resulted in changes of observed adenosine concentration after the scarification, pointing out that ENT1/2 have significant role in controlling extracellular concentration of this nucleoside. In order to identify possible regulation pathways for ENT, astrocytes where treated with adenosine, ATP, DPM and DPCPX, antagonist of A1 receptor. Results have shown that ATP upregulates both transporters as well as e-5NT, while adenosine enhances expression of ENT2 and diminishes expression of ENT1. Moreover, DPM and DPCPX have shown different effect on ENT1/2 expression, confirming that regulation of these two transporters depends on different pathways. Toward the end, we wanted to compare effects of mechanical injury to those caused by chemically induced hypoxia after 4-hour CoCl2 treatment, on activity of the components of adenosine system. Unlike the sratch wound injury, CoCl2 causes early upregulation of ENT2, e-5NT and HIF-1α, as well as much greater increase in extracellular adenosine concentration. Herein given results represent an overview of activity and expression the most important components of adenosine signaling system after the injury, therefore giving good foundation for future research of astrocyte role in control of adenosine in both physiological and pathological conditions, as well as for investigation of potential candidates for future TBI therapies.
Keywords:Astrocytes; Adenosine; Equilibrative nucleoside transporter; Traumatic brain injury; Ectonucleotidases; Cell culture; Purinergic signaling
Source:University of Belgrade, Faculty of Biology, 2013, 1-182
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