Activity of the astrocytes' adenosine signaling system components in model of traumatic brain injury in vivo and in vitro

Glavno obeležje traumatske povrede mozga (TPM) je reaktivna astroglioza koja, između ostalog, uzrokuje i promene u signalizaciji purinima. Posebno važan aspekt purinske signalizacije u patološkim procesima centralnog nervnog sistema predstavlja dinamika promena vanćelijskih koncentracija neuroprotektora adenozina. Stoga je cilj ove doktorske teze bio ispitivanje ekspresije i funkcije komponenti adenozinskog signalnog sistema astrocita u in vivo i in vitro modelu moždane povrede, sa posebnim osvrtom na ulogu ekvilibrišućih nukleozidnih transportera (ENT). U in vivo studiji, izvedenoj na modelu ubodne lezije kore prednjeg mozga pacova, je pokazano da povreda dovodi do dinamičnih promena u ekspresiji ENT, ektonukleotidaza i adenozinskog A1 receptora. Sem toga, povreda uzrokuje i ćelijsku re-distribuciju ENT1/2 i ushodnu regulaciju transportera na reaktivnim astrocitima, što je posebno izraženo sedmog dana nakon ozlede. Uloga astrocita u orkestraciji adenozinskog signalnog sistema nakon povrede je detaljnije ispitana in vitro, nakon skarifikacije astrocitnog jednosloja. Rezultati su pokazali da skarifikacija povećava ekspresiju ENT1 i ENT2 tek u kasnijim vremenima. Bifazna promena u ekspresiji ekto-5`-nukleotidaze (e-5NT) je iskazana prvobitnim smanjenjem i potom povećanjem ekspresije u kasnijim vremenima nakon skarifikacije. Pored toga, skarifikacija astrocitnog jednosloja vodi promenama u koncentracijama adenozina i njegovih metabolita u ćelijskom medijumu. Naime, porast koncentracija adenozina u ranim vremenima nakon povrede, bio je praćen padom u kasnijim vremenima. Blokiranje ENT dipiridamolom (DPM) je dovelo do promena u koncentracijama adenozina nakon skarifikacije, ukazavši na ulogu ENT1/2 u kontroli vanćelijskih koncentracija ovog nukleozida. Kako bi se nagovestili mogući putevi regulacije ENT, kultura astrocita je podvrgnuta tretmanima adenozinom, ATP, DPM i blokatorom A1 receptora DPCPX. Rezultati su pokazali da ATP ushodno reguliše oba transportera i e-5NT, dok adenozin povećava ekspresiju ENT2 a smanjuje ekspresiju ENT1. Takođe, drugačiji efekat na ekspresiju ENT1/2 su imali i DPM i DPCPX, potvrdivši da su ova dva transportera regulisana drugačijim putevima. Na kraju smo želeli da uporedimo efekte mehaničke povrede i CoCl2 izazvane hipoksije nakon četvoročasovnog na aktivnost komponenti adenozinskog sistema. Za razliku od skarifikacije, CoCl2 vodi ranom povećanju ekspresije ENT2, e-5NT i HIF-1α, kao i daleko većem povećanju vanćelijskih koncentracija adenozina. Dobijeni rezultati predstavljaju pregled eskpresije i aktivnosti najvažnijih komponenti adenozinskog sistema astrocita nakon povrede, dajući dobru osnovu za dalja ispitivanja uloge astrocita u kontroli kruženja adenozina u fiziološkim i patološkim stanjima, kao i potencijalne kandidate za buduće terapije TPM.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